- Home
- People
- Diversity
- Academics
- Seminars
- Bioinformatics Support
- Portal
Our research focuses on RNA polymerase, the central enzyme of gene expression in all free-living organisms. Our goal is to understand how RNA polymerase is regulated during the process of transcription (RNA synthesis). In organisms from bacteria to humans, the cell's ability to make long RNA chains, which include most mRNAs and some structural RNAs (e.g., rRNA), requires that extrinsic elongation regulators interact with RNA polymerase to suppress its innate tendency to fall into inactive off-line states that include long pauses, arrest, or termination. We seek to understand the fundamental properties of RNA polymerase that make it susceptible to pausing, arrest, or termination and how elongation regulators alter these properties. Additional research foci are the production of recombinant RNA polymerases from diverse bacterial lineages for antibiotic discovery and mechanistic dissection and the use of microbial synthetic biology for bioenergy applications.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
No abstract available.
[This corrects the article DOI: 10.1371/journal.pgen.1006372.].
The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.
The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression elements (promoters and ribosome-binding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate to ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression elements and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. We resolved current uncertainty about the functions of the Fe2+-dependent AdhB and Zn2+-dependent AdhA by showing that AdhB is preferred over AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimal E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.
The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.
Transcript termination is essential for accurate gene expression and the removal of RNA polymerase (RNAP) at the ends of transcription units. In bacteria, two mechanisms are responsible for proper transcript termination: intrinsic termination and Rho-dependent termination. Intrinsic termination is mediated by signals directly encoded within the DNA template and nascent RNA, whereas Rho-dependent termination relies upon the adenosine triphosphate-dependent RNA translocase Rho, which binds nascent RNA and dissociates the elongation complex. Although significant progress has been made in understanding these pathways, fundamental details remain undetermined. Among those that remain unresolved are the existence of an inactivated intermediate in the intrinsic termination pathway, the role of Rho-RNAP interactions in Rho-dependent termination, and the mechanisms by which accessory factors and nucleoid-associated proteins affect termination. We describe current knowledge, discuss key outstanding questions, and highlight the importance of defining the structural rearrangements of RNAP that are involved in the two mechanisms of transcript termination.
The vectorial (5'-to-3' at varying velocity) synthesis of RNA by cellular RNA polymerases (RNAPs) creates a rugged kinetic landscape, demarcated by frequent, sometimes long-lived, pauses. In addition to myriad gene-regulatory roles, these pauses temporally and spatially program the co-transcriptional, hierarchical folding of biologically active RNAs. Conversely, these RNA structures, which form inside or near the RNA exit channel, interact with the polymerase and adjacent protein factors to influence RNA synthesis by modulating pausing, termination, antitermination, and slippage. Here, we review the evolutionary origin, mechanistic underpinnings, and regulatory consequences of this interplay between RNAP and nascent RNA structure. We categorize and rationalize the extensive linkage between the transcriptional machinery and its product, and provide a framework for future studies.
Imidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries. However, residual IILs are toxic to fermentative microbes such as Saccharomyces cerevisiae, making IIL-tolerance a key property for strain engineering. To enable rational engineering, we used chemical genomic profiling to understand the effects of IILs on S. cerevisiae. We found that IILs likely target mitochondria as their chemical genomic profiles closely resembled that of the mitochondrial membrane disrupting agent valinomycin. Further, several deletions of genes encoding mitochondrial proteins exhibited increased sensitivity to IIL. High-throughput chemical proteomics confirmed effects of IILs on mitochondrial protein levels. IILs induced abnormal mitochondrial morphology, as well as altered polarization of mitochondrial membrane potential similar to valinomycin. Deletion of the putative serine/threonine kinase PTK2 thought to activate the plasma-membrane proton efflux pump Pma1p conferred a significant IIL-fitness advantage. Conversely, overexpression of PMA1 conferred sensitivity to IILs, suggesting that hydrogen ion efflux may be coupled to influx of the toxic imidazolium cation. PTK2 deletion conferred resistance to multiple IILs, including [EMIM]Cl, [BMIM]Cl, and [EMIM]Ac. An engineered, xylose-converting ptk2∆ S. cerevisiae (Y133-IIL) strain consumed glucose and xylose faster and produced more ethanol in the presence of 1 % [BMIM]Cl than the wild-type PTK2 strain. We propose a model of IIL toxicity and resistance. This work demonstrates the utility of chemical genomics-guided biodesign for development of superior microbial biocatalysts for the ever-changing landscape of fermentation inhibitors.
Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders E. coli more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene ref. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to E. coli in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens Neisseria gonorrhoeae and Staphylococcus aureus serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence.
Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ(70), can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ(70) retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ(70)-RNAP complex during initiation from the λ PR' promoter and throughout the elongation of a long (>2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound σ(70) and the kinetics of σ(70) release from actively transcribing complexes. σ(70) release from mature elongation complexes was slow (0.0038 s(-1)); a substantial subpopulation of elongation complexes retained σ(70) throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ(70) manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of σ(70) during transcription.
Microbial conversion of lignocellulosic feedstocks into biofuels remains an attractive means to produce sustainable energy. It is essential to produce lignocellulosic hydrolysates in a consistent manner in order to study microbial performance in different feedstock hydrolysates. Because of the potential to introduce microbial contamination from the untreated biomass or at various points during the process, it can be difficult to control sterility during hydrolysate production. In this study, we compared hydrolysates produced from AFEX-pretreated corn stover and switchgrass using two different methods to control contamination: either by autoclaving the pretreated feedstocks prior to enzymatic hydrolysis, or by introducing antibiotics during the hydrolysis of non-autoclaved feedstocks. We then performed extensive chemical analysis, chemical genomics, and comparative fermentations to evaluate any differences between these two different methods used for producing corn stover and switchgrass hydrolysates. Autoclaving the pretreated feedstocks could eliminate the contamination for a variety of feedstocks, whereas the antibiotic gentamicin was unable to control contamination consistently during hydrolysis. Compared to the addition of gentamicin, autoclaving of biomass before hydrolysis had a minimal effect on mineral concentrations, and showed no significant effect on the two major sugars (glucose and xylose) found in these hydrolysates. However, autoclaving elevated the concentration of some furanic and phenolic compounds. Chemical genomics analyses using Saccharomyces cerevisiae strains indicated a high correlation between the AFEX-pretreated hydrolysates produced using these two methods within the same feedstock, indicating minimal differences between the autoclaving and antibiotic methods. Comparative fermentations with S. cerevisiae and Zymomonas mobilis also showed that autoclaving the AFEX-pretreated feedstocks had no significant effects on microbial performance in these hydrolysates. Our results showed that autoclaving the pretreated feedstocks offered advantages over the addition of antibiotics for hydrolysate production. The autoclaving method produced a more consistent quality of hydrolysate, and also showed negligible effects on microbial performance. Although the levels of some of the lignocellulose degradation inhibitors were elevated by autoclaving the feedstocks prior to enzymatic hydrolysis, no significant effects on cell growth, sugar utilization, or ethanol production were seen during bacterial or yeast fermentations in hydrolysates produced using the two different methods.
Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstream of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σ(A) dissociation.
RNA polymerase inhibitors like the CBR class that target the enzyme's complex catalytic center are attractive leads for new antimicrobials. Catalysis by RNA polymerase involves multiple rearrangements of bridge helix, trigger loop, and active-center side chains that isomerize the triphosphate of bound NTP and two Mg(2+) ions from a preinsertion state to a reactive configuration. CBR inhibitors target a crevice between the N-terminal portion of the bridge helix and a surrounding cap region within which the bridge helix is thought to rearrange during the nucleotide addition cycle. We report crystal structures of CBR inhibitor/Escherichia coli RNA polymerase complexes as well as biochemical tests that establish two distinct effects of the inhibitors on the RNA polymerase catalytic site. One effect involves inhibition of trigger-loop folding via the F loop in the cap, which affects both nucleotide addition and hydrolysis of 3'-terminal dinucleotides in certain backtracked complexes. The second effect is trigger-loop independent, affects only nucleotide addition and pyrophosphorolysis, and may involve inhibition of bridge-helix movements that facilitate reactive triphosphate alignment.
Histone-like nucleoid structuring (H-NS) protein is a component of bacterial chromatin and influences gene expression both locally and on a global scale. Although H-NS is broadly considered a silencer of transcription, the mechanisms by which H-NS inhibits gene expression remain poorly understood. Here we discuss recent advances in the context of a 'love-hate' relationship between H-NS and RNA polymerase, in which these factors recognise similar DNA sequences but interfere with each other's activity. Understanding the complex relationship between H-NS and RNA polymerase may unite the multiple models that have been proposed to describe gene silencing by H-NS.
A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides. We report the discovery of poacic acid, a potent antifungal compound found in lignocellulosic hydrolysates of grasses. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. Morphological analysis revealed that cells treated with poacic acid behaved similarly to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Poacic acid causes rapid cell lysis and is synergistic with caspofungin and fluconazole. The cellular target was identified; poacic acid localized to the cell wall and inhibited β-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding β-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development. The discovery of poacic acid as a natural antifungal agent targeting β-1,3-glucan highlights the potential side use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds and further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates.
Bacterial H-NS forms nucleoprotein filaments that spread on DNA and bridge distant DNA sites. H-NS filaments co-localize with sites of Rho-dependent termination in Escherichia coli, but their direct effects on transcriptional pausing and termination are untested. In this study, we report that bridged H-NS filaments strongly increase pausing by E. coli RNA polymerase at a subset of pause sites with high potential for backtracking. Bridged but not linear H-NS filaments promoted Rho-dependent termination by increasing pause dwell times and the kinetic window for Rho action. By observing single H-NS filaments and elongating RNA polymerase molecules using atomic force microscopy, we established that bridged filaments surround paused complexes. Our results favor a model in which H-NS-constrained changes in DNA supercoiling driven by transcription promote pausing at backtracking-susceptible sites. Our findings provide a mechanistic rationale for H-NS stimulation of Rho-dependent termination in horizontally transferred genes and during pervasive antisense and noncoding transcription in bacteria.
CarD is an essential mycobacterial protein that binds the RNA polymerase (RNAP) and affects the transcriptional profile of Mycobacterium smegmatis and Mycobacterium tuberculosis (6). We predicted that CarD was directly regulating RNAP function but our prior experiments had not determined at what stage of transcription CarD was functioning and at which genes CarD interacted with the RNAP. To begin to address these open questions, we performed Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. The distribution of RNAP subunits β and σ(A) were also profiled. We expected that RNAP β would be present throughout transcribed regions and RNAP σ(A) would be predominantly enriched at promoters based on work in Escherichia coli (3), however this had yet to be determined in mycobacteria. The ChIP-seq analyses revealed that CarD was never present on the genome in the absence of RNAP, was primarily associated with promoter regions, and was highly correlated with the distribution of RNAP σ(A). The colocalization of σ(A) and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. Here we describe in detail the data from the ChIP-seq experiments associated with the study published by Srivastava and colleagues in the Proceedings of the National Academy of Science in 2013 (5) as well as discuss the findings from this dataset in relation to both CarD and mycobacterial transcription as a whole. The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE48164).
The conformational dynamics of the polymorphous trigger loop (TL) in RNA polymerase (RNAP) underlie multiple steps in the nucleotide addition cycle and diverse regulatory mechanisms. These mechanisms include nascent RNA hairpin-stabilized pausing, which inhibits TL folding into the trigger helices (TH) required for rapid nucleotide addition. The nascent RNA pause hairpin forms in the RNA exit channel and promotes opening of the RNAP clamp domain, which in turn stabilizes a partially folded, paused TL conformation that disfavors TH formation. We report that inhibiting TH unfolding with a disulfide crosslink slowed multiround nucleotide addition only modestly but eliminated hairpin-stabilized pausing. Conversely, a substitution that disrupts the TH folding pathway and uncouples establishment of key TH-NTP contacts from complete TH formation and clamp movement allowed rapid catalysis and eliminated hairpin-stabilized pausing. We also report that the active-site distal arm of the TH aids TL folding, but that a 188-aa insertion in the Escherichia coli TL (sequence insertion 3; SI3) disfavors TH formation and stimulates pausing. The effect of SI3 depends on the jaw domain, but not on downstream duplex DNA. Our results support the view that both SI3 and the pause hairpin modulate TL folding in a constrained pathway of intermediate states.
The rates of RNA synthesis and the folding of nascent RNA into biologically active structures are linked via pausing by RNA polymerase (RNAP). Structures that form within the RNA-exit channel can either increase pausing by interacting with RNAP or decrease pausing by preventing backtracking. Conversely, pausing is required for proper folding of some RNAs. Opening of the RNAP clamp domain has been proposed to mediate some effects of nascent-RNA structures. However, the connections among RNA structure formation and RNAP clamp movement and catalytic activity remain uncertain. Here, we assayed exit-channel structure formation in Escherichia coli RNAP with disulfide cross-links that favor closed- or open-clamp conformations and found that clamp position directly influences RNA structure formation and RNAP catalytic activity. We report that exit-channel RNA structures slow pause escape by favoring clamp opening through interactions with the flap that slow translocation.
Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5-hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts.
The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting S. cerevisiae strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent S. cerevisiae strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in GRE3, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.
The molecular mechanisms of ethanol toxicity and tolerance in bacteria, although important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and have revealed multiple mechanisms of tolerance, but it remains difficult to separate the direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, and then characterized mechanisms of toxicity and resistance using genome-scale DNAseq, RNAseq, and ribosome profiling coupled with specific assays of ribosome and RNA polymerase function. Evolved alleles of metJ, rho, and rpsQ recapitulated most of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. Ethanol induced miscoding errors during protein synthesis, from which the evolved rpsQ allele protected cells by increasing ribosome accuracy. Ribosome profiling and RNAseq analyses established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis through direct effects on ribosomes and RNA polymerase conformations are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ help protect these central dogma processes in the presence of ethanol.
Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse regulatory roles. Although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. Using nascent transcript sequencing, we identified a 16-nucleotide consensus pause sequence in Escherichia coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses demonstrate that these pauses result from RNAP-nucleic acid interactions that inhibit next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from diverse lineages and is enriched at translation start sites in both E. coli and Bacillus subtilis. Our results thus reveal a conserved mechanism unifying known and newly identified pause events.
Intrinsic terminators, which encode GC-rich RNA hairpins followed immediately by a 7-to-9-nucleotide (nt) U-rich "U-tract," play principal roles of punctuating and regulating transcription in most bacteria. However, canonical intrinsic terminators with strong U-tracts are underrepresented in some bacterial lineages, notably mycobacteria, leading to proposals that their RNA polymerases stop at noncanonical intrinsic terminators encoding various RNA structures lacking U-tracts. We generated recombinant forms of mycobacterial RNA polymerase and its major elongation factors NusA and NusG to characterize mycobacterial intrinsic termination. Using in vitro transcription assays devoid of possible mycobacterial contaminants, we established that mycobacterial RNA polymerase terminates more efficiently than Escherichia coli RNA polymerase at canonical terminators with imperfect U-tracts but does not terminate at putative terminators lacking U-tracts even in the presence of mycobacterial NusA and NusG. However, mycobacterial NusG exhibits a novel termination-stimulating activity that may allow intrinsic terminators with suboptimal U-tracts to function efficiently. IMPORTANCE Bacteria rely on transcription termination to define and regulate units of gene expression. In most bacteria, precise termination and much regulation by attenuation are accomplished by intrinsic terminators that encode GC-rich hairpins and U-tracts necessary to disrupt stable transcription elongation complexes. Thus, the apparent dearth of canonical intrinsic terminators with recognizable U-tracts in mycobacteria is of significant interest both because noncanonical intrinsic terminators could reveal novel routes to destabilize transcription complexes and because accurate understanding of termination is crucial for strategies to combat mycobacterial diseases and for computational bioinformatics generally. Our finding that mycobacterial RNA polymerase requires U-tracts for intrinsic termination, which can be aided by NusG, will guide future study of mycobacterial transcription and aid improvement of predictive algorithms to annotate bacterial genome sequences.
Lignocellulosic hydrolysate (LCH) inhibitors are a large class of bioactive molecules that arise from pretreatment, hydrolysis, and fermentation of plant biomass. These diverse compounds reduce lignocellulosic biofuel yields by inhibiting cellular processes and diverting energy into cellular responses. LCH inhibitors present one of the most significant challenges to efficient biofuel production by microbes. Development of new strains that lessen the effects of LCH inhibitors is an economically favorable strategy relative to expensive detoxification methods that also can reduce sugar content in deconstructed biomass. Systems biology analyses and metabolic modeling combined with directed evolution and synthetic biology are successful strategies for biocatalyst development, and methods that leverage state-of-the-art tools are needed to overcome inhibitors more completely. This perspective considers the energetic costs of LCH inhibitors and technologies that can be used to overcome their drain on conversion efficiency. We suggest academic and commercial research groups could benefit by sharing data on LCH inhibitors and implementing "translational biofuel research."
In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo.
Transcript elongation by bacterial RNA polymerase (RNAP) is thought to be regulated at pause sites by open versus closed positions of the RNAP clamp domain, pause-suppressing regulators like NusG and RfaH that stabilize the closed-clampRNAP conformation, and pause-enhancing regulators like NusA and exit channel nascent RNA structures that stabilize the open clamp RNAP conformation. However, the mutual effects of these protein and RNA regulators on RNAP conformation are incompletely understood. For example, it is unknown whether NusA directly interacts with exit channel duplexes and whether formation of exit channel duplexes and RfaH binding compete by favoring the open and closed RNAP conformations. We report new insights into these mechanisms using antisense oligonucleotide mimics of a pause RNA hairpin from the leader region of the his biosynthetic operon of enteric bacteria like Escherichia coli. By systematically varying the structure and length of the oligonucleotide mimic, we determined that full pause stabilization requires an RNA-RNA duplex of at least 8 bp or a DNA-RNA duplex of at least 11 bp; RNA-RNA duplexes were more effective than DNA-RNA. NusA stimulation of pausing was optimal with 10-bp RNA-RNA duplexes and was aided by single-stranded RNA upstream of the duplex but was significantly reduced with DNA-RNA duplexes. Our results favor direct NusA stabilization of exit channel duplexes, which consequently affect RNAP clamp conformation. Effects of RfaH, which suppresses oligo-stabilization of pausing, were competitive with antisense oligonucleotide concentration, suggesting that RfaH and exit channel duplexes compete via opposing effects on RNAP clamp conformation.
Despite the importance of maintaining redox homeostasis for cellular viability, how cells control redox balance globally is poorly understood. Here we provide new mechanistic insight into how the balance between reduced and oxidized electron carriers is regulated at the level of gene expression by mapping the regulon of the response regulator ArcA from Escherichia coli, which responds to the quinone/quinol redox couple via its membrane-bound sensor kinase, ArcB. Our genome-wide analysis reveals that ArcA reprograms metabolism under anaerobic conditions such that carbon oxidation pathways that recycle redox carriers via respiration are transcriptionally repressed by ArcA. We propose that this strategy favors use of catabolic pathways that recycle redox carriers via fermentation akin to lactate production in mammalian cells. Unexpectedly, bioinformatic analysis of the sequences bound by ArcA in ChIP-seq revealed that most ArcA binding sites contain additional direct repeat elements beyond the two required for binding an ArcA dimer. DNase I footprinting assays suggest that non-canonical arrangements of cis-regulatory modules dictate both the length and concentration-sensitive occupancy of DNA sites. We propose that this plasticity in ArcA binding site architecture provides both an efficient means of encoding binding sites for ArcA, σ(70)-RNAP and perhaps other transcription factors within the same narrow sequence space and an effective mechanism for global control of carbon metabolism to maintain redox homeostasis.
Chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) has been successfully used for genome-wide profiling of transcription factor binding sites, histone modifications, and nucleosome occupancy in many model organisms and humans. Because the compact genomes of prokaryotes harbor many binding sites separated by only few base pairs, applications of ChIP-Seq in this domain have not reached their full potential. Applications in prokaryotic genomes are further hampered by the fact that well studied data analysis methods for ChIP-Seq do not result in a resolution required for deciphering the locations of nearby binding events. We generated single-end tag (SET) and paired-end tag (PET) ChIP-Seq data for σâ
Many pathogenic E. coli strains secrete virulence factors using type II secretory systems, homologs of which are widespread in Gram-negative bacteria. Recently, the enteropathogenic Escherichia coli strain E2348/69 was shown to secrete and surface-anchor SslE, a biofilm-promoting virulence factor, via a type II secretion system. Genes encoding SslE and its associated secretion system are conserved in some non-pathogenic E. coli, including the commonly-used W (Waksman) strain. We report here that E. coli W uses its type II secretion system to export a cognate SslE protein. SslE secretion is temperature- and nutrient-dependent, being robust at 37°C in rich medium but strongly repressed by lower temperatures or nutrient limitation. Fusing either of two glycosyl hydrolases to the C-terminus of SslE prevented it from being secreted or surface-exposed. We screened mutations that inactivated the type II secretion system for stress-related phenotypes and found that inactivation of the secretion system conferred a modest increase in tolerance to high concentrations of urea. Additionally, we note that the genes encoding this secretion system are present at a hypervariable locus and have been independently lost or gained in different lineages of E. coli. The non-pathogenic E. coli W strain shares the extracellular virulence factor SslE, and its associated secretory system, with pathogenic E. coli strains. The pattern of regulation of SslE secretion we observed suggests that SslE plays a role in colonization of mammalian hosts by non-pathogenic as well as pathogenic E. coli. Our work provides a non-pathogenic model system for the study of SslE secretion, and informs future research into the function of SslE during host colonization.
Transcriptional pausing, which regulates transcript elongation in both prokaryotes and eukaryotes, is thought to involve formation of alternative RNA polymerase conformations in which nucleotide addition is inhibited in part by restriction of trigger loop (TL) folding. The polymorphous TL must convert from a random coil to a helical hairpin that contacts the nucleotide triphosphate (NTP) substrate to allow rapid nucleotide addition. Understanding the distribution of TL conformations in different enzyme states is made difficult by the TL's small size and sensitive energetics. Here, we report a Cys-pair reporter strategy to elucidate the relative occupancies of different TL conformations in E. coli RNA polymerase based on the ability of Cys residues engineered into the TL and surrounding regions to form disulfide bonds. Our results indicate that a paused complex stabilized by a nascent RNA hairpin favors nonproductive TL conformations that persist after NTP binding but can be reversed by the elongation factor RfaH.
No abstract available.
FNR is a well-studied global regulator of anaerobiosis, which is widely conserved across bacteria. Despite the importance of FNR and anaerobiosis in microbial lifestyles, the factors that influence its function on a genome-wide scale are poorly understood. Here, we report a functional genomic analysis of FNR action. We find that FNR occupancy at many target sites is strongly influenced by nucleoid-associated proteins (NAPs) that restrict access to many FNR binding sites. At a genome-wide level, only a subset of predicted FNR binding sites were bound under anaerobic fermentative conditions and many appeared to be masked by the NAPs H-NS, IHF and Fis. Similar assays in cells lacking H-NS and its paralog StpA showed increased FNR occupancy at sites bound by H-NS in WT strains, indicating that large regions of the genome are not readily accessible for FNR binding. Genome accessibility may also explain our finding that genome-wide FNR occupancy did not correlate with the match to consensus at binding sites, suggesting that significant variation in ChIP signal was attributable to cross-linking or immunoprecipitation efficiency rather than differences in binding affinities for FNR sites. Correlation of FNR ChIP-seq peaks with transcriptomic data showed that less than half of the FNR-regulated operons could be attributed to direct FNR binding. Conversely, FNR bound some promoters without regulating expression presumably requiring changes in activity of condition-specific transcription factors. Such combinatorial regulation may allow Escherichia coli to respond rapidly to environmental changes and confer an ecological advantage in the anaerobic but nutrient-fluctuating environment of the mammalian gut.
CarD, an essential transcription regulator in Mycobacterium tuberculosis, directly interacts with the RNA polymerase (RNAP). We used a combination of in vivo and in vitro approaches to establish that CarD is a global regulator that stimulates the formation of RNAP-holoenzyme open promoter (RPo) complexes. We determined the X-ray crystal structure of Thermus thermophilus CarD, allowing us to generate a structural model of the CarD/RPo complex. On the basis of our structural and functional analyses, we propose that CarD functions by forming protein/protein and protein/DNA interactions that bridge the RNAP to the promoter DNA. CarD appears poised to interact with a DNA structure uniquely presented by the RPo: the splayed minor groove at the double-stranded/single-stranded DNA junction at the upstream edge of the transcription bubble. Thus, CarD uses an unusual mechanism for regulating transcription, sensing the DNA conformation where transcription bubble formation initiates.
Transcriptional pausing by multisubunit RNA polymerases (RNAPs) is a key mechanism for regulating gene expression in both prokaryotes and eukaryotes and is a prerequisite for transcription termination. Pausing and termination states are thought to arise through a common, elemental pause state that is inhibitory for nucleotide addition. We report three crystal structures of Thermus RNAP elemental paused elongation complexes (ePECs). The structures reveal the same relaxed, open-clamp RNAP conformation in the ePEC that may arise by failure to re-establish DNA contacts during translocation. A kinked bridge-helix sterically blocks the RNAP active site, explaining how this conformation inhibits RNAP catalytic activity. Our results provide a framework for understanding how RNA hairpin formation stabilizes the paused state and how the ePEC intermediate facilitates termination.
Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global colocalization of the histone-like nucleoid-structuring protein (H-NS) with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes.
Rho termination factor is an essential hexameric helicase responsible for terminating 20-50% of all mRNA synthesis in Escherichia coli. We used single-molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho utilization site of the λtR1 terminator. Our results are consistent with Rho complexes adopting two states: one that binds 57 ± 2nt of RNA across all six of the Rho primary binding sites, and another that binds 85 ± 2nt at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5'→3' toward RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the Rho utilization site and RNAP. These findings lead to a general model for Rho binding and translocation and establish a novel experimental approach that should facilitate additional single-molecule studies of RNA-binding proteins.
Single-molecule studies of RNA polymerase II (RNAP II) require high yields of transcription elongation complexes (TECs) with long DNA tethers upstream and downstream of the TEC. Here we report on a robust system to reconstitute both yeast and mammalian RNAP II with an efficiency of ~80% into TECs that elongate with an efficiency of ~90%, followed by rapid, high-efficiency tripartite ligation of long DNA fragments upstream and downstream of the reconstituted TECs. Single mammalian and yeast TECs reconstituted with this method have been successfully used in an optical-trapping transcription assay capable of applying forces that either assist or hinder transcript elongation.
NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a β barrel in NusG and an α hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here, we show that the released RfaH-CTD completely refolds from an all-α to an all-β conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated. PAPERFLICK:
The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.
During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre- or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate.
Using a fluorescence method called colocalization single-molecule spectroscopy (CoSMoS), Friedman and Gelles dissect the kinetics of transcription initiation at a bacterial promoter. Ultimately, CoSMoS could greatly aid the study of the effects of DNA sequence and transcription factors on both prokaryotic and eukaryotic promoters.
Transcriptional pausing by RNA polymerase (RNAP) plays an essential role in gene regulation. Pausing is modified by various elongation factors, including prokaryotic NusA, but the mechanisms underlying pausing and NusA function remain unclear. Alternative models for pausing invoke blockade events that precede translocation (on-pathway), enzyme backtracking (off-pathway), or isomerization to a nonbacktracked, elemental pause state (off-pathway). We employed an optical trapping assay to probe the motions of individual RNAP molecules transcribing a DNA template carrying tandem repeats encoding the his pause, subjecting these enzymes to controlled forces. NusA significantly decreased the pause-free elongation rate of RNAP while increasing the probability of entry into short- and long-lifetime pauses, in a manner equivalent to exerting a ~19 pN force opposing transcription. The effects of force and NusA on pause probabilities and lifetimes support a reaction scheme where nonbacktracked, elemental pauses branch off the elongation pathway from the pretranslocated state of RNAP.
The process of transcription termination is essential to proper expression of bacterial genes and, in many cases, to the regulation of bacterial gene expression. Two types of bacterial transcriptional terminators are known to control gene expression. Intrinsic terminators dissociate transcription complexes without the assistance of auxiliary factors. Rho-dependent terminators are sites of dissociation mediated by an RNA helicase called Rho. Despite decades of study, the molecular mechanisms of both intrinsic and Rho-dependent termination remain uncertain in key details. Most knowledge is based on the study of a small number of model terminators. The extent of sequence diversity among functional terminators and the extent of mechanistic variation as a function of sequence diversity are largely unknown. In this review, we consider the current state of knowledge about bacterial termination mechanisms and the relationship between terminator sequence and steps in the termination mechanism.
The flap domain of multisubunit RNA polymerases (RNAPs), also called the wall, forms one side of the RNA exit channel. In bacterial RNAP, the mobile part of the flap is called the flap tip and makes essential contacts with initiation and elongation factors. Cocrystal structures suggest that the orthologous part of eukaryotic RNAPII, called the flap loop, contacts transcription factor IIB (TFIIB), but the function of the flap loop has not been assessed. We constructed and tested a deletion of the flap loop in human RNAPII (subunit RPB2 Δ873-884) that removes the flap loop interaction interface with TFIIB. Genome-wide analysis of the distribution of the RNAPII with the flap loop deletion expressed in a human embryonic kidney cell line (HEK 293) revealed no effect of the flap loop on global transcription initiation, RNAPII occupancy within genes, or the efficiency of promoter escape and productive elongation. In vitro, the flap loop deletion had no effect on promoter binding, abortive initiation or promoter escape, TFIIS-stimulated transcript cleavage, or inhibition of transcript elongation by the complex of negative elongation factor (NELF) and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF). A modest effect on transcript elongation and pausing was suppressed by TFIIF. Although similar to the flap tip of bacterial RNAP, the RNAPII flap loop is not equivalently essential.
Translocation of RNA polymerase on DNA is thought to involve oscillations between pretranslocated and posttranslocated states that are rectified by nucleotide addition or pyrophosphorolysis. The pretranslocated register is also a precursor to transcriptional pause states that mediate regulation of transcript elongation. However, the determinants of bias between the pretranslocated and posttranslocated states are incompletely understood. To investigate translocation bias in multisubunit RNA polymerases, we measured rates of pyrophosphorolysis, which occurs in the pretranslocated register, in minimal elongation complexes containing T. thermophilus or E. coli RNA polymerase. Our results suggest that the identity of RNA:DNA nucleotides in the active site are strong determinants of susceptibility to pyrophosphorolysis, and thus translocation bias, with the 3' RNA nucleotide favoring the pretranslocated state in the order U > C > A > G. The preference of 3' U vs G for the pretranslocated register appeared to be universal among both bacterial and eukaryotic RNA polymerases and was confirmed by exonuclease III footprinting of defined elongation complexes. However, the relationship of pyrophosphate concentration to the rate of pyrophosphorolysis of 3' U-containing versus 3' G-containing elongation complexes did not match predictions of a simple mechanism in which 3'-RNA seqeunce affects only translocation bias and pyrophosphate (PPi) binds only to the pretranslocated state.
In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the β gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the β' clamp helices. We hypothesize that upon binding to the clamp helices and the gate loop RfaH bridges the gap across the DNA channel, stabilizing RNAP contacts with nucleic acid and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity.
The ability to examine gene regulation in living cells has been greatly enabled by the development of chromatin immunoprecipitation (ChIP) methodology. ChIP captures a snapshot of protein-DNA interactions in vivo and has been used to study interactions in bacteria, yeast, and mammalian cell culture. ChIP conditions vary depending upon the organism and the nature of the DNA-binding proteins under study. Here, we describe a customized ChIP protocol to examine the genome-wide distribution of a mobile DNA-binding enzyme, Escherichia coli RNA Polymerase (RNAP) as well as the factors that dynamically associate with RNAP during different stages of transcription. We describe new data analysis methods for determining the association of a broadly distributed DNA-binding complex. Further, we describe our approach of combining small molecules and antibiotics that perturb specific cellular events with ChIP and genomic platforms to dissect mechanisms of gene regulation in vivo. The chemical genomic methods can be leveraged to map natural and cryptic promoters and transcription units, annotate genomes, and reveal coupling between different processes in regulation of genes. This approach provides the framework for engineering gene networks and controlling biological output in a desired manner.
Transcription is the first of many biochemical steps that turn the genetic information found in DNA into the proteins responsible for driving cellular processes. In this review, we highlight certain advantages of single-molecule techniques in the study of prokaryotic and eukaryotic transcription, and the specific ways in which these techniques complement conventional, ensemble-based biochemistry. We focus on recent literature, highlighting examples where single-molecule methods have provided fresh insights into mechanism. We also present recent technological advances and outline future directions in the field.
Genes must be stably integrated into bacterial chromosomes for complementation of gene deletion mutants in animal infection experiments or to express antigens in vaccine strains. However, with currently available vectors it is cumbersome to create multiple, stable, unmarked chromosomal integrations in mycobacteria. Here, we have constructed a novel integration vector for mycobacteria that enables expression of genes from a cassette protected from transcriptional interference by bi-directional transcriptional terminators proven to be highly efficient in in vitro transcription termination assays. Removal of the integrase gene by a site-specific recombinase, easily identifiable by loss of a backbone reporter gene, stabilizes the integration cassette and makes this vector ideally suitable for infection experiments. This integration vector can be easily adapted to different mycobacteriophage attachment sites (attB) due to its modular design. Integration of a gfp expression cassette at the L5, Giles and Ms6 attB sites in the chromosomes of Mycobacterium smegmatis and Mycobacterium tuberculosis yielded identical gfp expression levels, indicating that none of these sites are compromised for gene expression. The copy number of pAL5000-based extrachromosomal plasmids is 23 in M. smegmatis as determined by quantitative real-time PCR and accounts for the previously observed drastic reduction of gene expression upon integration of plasmids into the chromosome of mycobacteria. Gfp expression and fluorescence of M. smegmatis and M. tuberculosis strains with multiple integrations of gfp increased concomitantly with the copy number demonstrating that these vectors can be used to generate stronger phenotypes and/or to analyze several genes simultaneously in vivo.
Specific small deletions within the rpoC gene encoding the β'-subunit of RNA polymerase (RNAP) are found repeatedly after adaptation of Escherichia coli K-12 MG1655 to growth in minimal media. Here we present a multiscale analysis of these mutations. At the physiological level, the mutants grow 60% faster than the parent strain and convert the carbon source 15-35% more efficiently to biomass, but grow about 30% slower than the parent strain in rich medium. At the molecular level, the kinetic parameters of the mutated RNAP were found to be altered, resulting in a 4- to 30-fold decrease in open complex longevity at an rRNA promoter and a ∼10-fold decrease in transcriptional pausing, with consequent increase in transcript elongation rate. At a genome-scale, systems biology level, gene expression changes between the parent strain and adapted RNAP mutants reveal large-scale systematic transcriptional changes that influence specific cellular processes, including strong down-regulation of motility, acid resistance, fimbria, and curlin genes. RNAP genome-binding maps reveal redistribution of RNAP that may facilitate relief of a metabolic bottleneck to growth. These findings suggest that reprogramming the kinetic parameters of RNAP through specific mutations allows regulatory adaptation for optimal growth in new environments.
The RNA polymerase 'bridge helix' is a metastable α-helix that spans the leading edge of the enzyme active-site cleft. A new study published in BMC Biology reveals surprising tolerance to helix-disrupting changes in a region previously thought crucial for translocation, and suggests roles for two hinge-like segments of the bridge helix in coordinating modules that move during the nucleotide-addition cycle.
NusA is a core, multidomain regulator of transcript elongation in bacteria and archaea. Bacterial NusA interacts with elongating complexes and the nascent RNA transcript in ways that stimulate pausing and termination but that can be switched to antipausing and antitermination by other accessory proteins. This regulatory complexity of NusA likely depends on its multidomain structure, but it remains unclear which NusA domains possess which regulatory activity and how they interact with elongating RNA polymerase. We used a series of truncated NusA proteins to measure the effect of the NusA domains on transcriptional pausing and termination. We find that the N-terminal domain (NTD) of NusA is necessary and sufficient for enhancement of transcriptional pausing and that the other NusA domains contribute to NusA binding to elongating complexes. Stimulation of intrinsic termination requires higher concentrations of NusA and involves both the NTD and other NusA domains. Using a tethered chemical protease in addition to protein-RNA cross-linking, we show that the NusA NTD contacts the RNA exit channel of RNA polymerase. Finally, we report evidence that the NusA NTD recognizes duplex RNA in the RNA exit channel.
The Escherichia coli transcription system is the best characterized from a biochemical and genetic point of view and has served as a model system. Nevertheless, a molecular understanding of the details of E. coli transcription and its regulation, and therefore its full exploitation as a model system, has been hampered by the absence of high-resolution structural information on E. coli RNA polymerase (RNAP). We use a combination of approaches, including high-resolution X-ray crystallography, ab initio structural prediction, homology modeling, and single-particle cryo-electron microscopy, to generate complete atomic models of E. coli core RNAP and an E. coli RNAP ternary elongation complex. The detailed and comprehensive structural descriptions can be used to help interpret previous biochemical and genetic data in a new light and provide a structural framework for designing experiments to understand the function of the E. coli lineage-specific insertions and their role in the E. coli transcription program.
We report observations suggesting that the transcription elongation factor NusA promotes a previously unrecognized class of transcription-coupled repair (TCR) in addition to its previously proposed role in recruiting translesion synthesis (TLS) DNA polymerases to gaps encountered during transcription. Earlier, we reported that NusA physically and genetically interacts with the TLS DNA polymerase DinB (DNA pol IV). We find that Escherichia coli nusA11(ts) mutant strains, at the permissive temperature, are highly sensitive to nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide but not to UV radiation. Gene expression profiling suggests that this sensitivity is unlikely to be due to an indirect effect on gene expression affecting a known DNA repair or damage tolerance pathway. We demonstrate that an N(2)-furfuryl-dG (N(2)-f-dG) lesion, a structural analog of the principal lesion generated by NFZ, blocks transcription by E. coli RNA polymerase (RNAP) when present in the transcribed strand, but not when present in the nontranscribed strand. Our genetic analysis suggests that NusA participates in a nucleotide excision repair (NER)-dependent process to promote NFZ resistance. We provide evidence that transcription plays a role in the repair of NFZ-induced lesions through the isolation of RNAP mutants that display altered ability to survive NFZ exposure. We propose that NusA participates in an alternative class of TCR involved in the identification and removal of a class of lesion, such as the N(2)-f-dG lesion, which are accurately and efficiently bypassed by DinB in addition to recruiting DinB for TLS at gaps encountered by RNAP.
NusG is an essential transcription factor in Escherichia coli that is capable of increasing the overall rate of transcription. Transcript elongation by RNA polymerase (RNAP) is frequently interrupted by pauses of varying durations, and NusG is known to decrease the occupancy of at least some paused states. However, it has not been established whether NusG enhances transcription chiefly by (1) increasing the rate of elongation between pauses, (2) reducing the lifetimes of pauses, or (3) reducing the rate of entry into paused states. Here, we studied transcription by single molecules of RNAP under various conditions of ribonucleoside triphosphate concentration, applied load, and temperature, using an optical trapping assay capable of distinguishing pauses as brief as 1 s. We found that NusG increases the rate of elongation, that is, the pause-free velocity along the template. Because pauses are off-pathway states that compete with elongation, we observed a concomitant decrease in the rate of entry into short-lifetime, paused states. The effects on short pauses and elongation were comparatively modest, however. More dramatic was the effect of NusG on suppressing entry into long-lifetime ("stabilized") pauses. Because a significant fraction of the time required for the transcription of a typical gene may be occupied by long pauses, NusG is capable of exerting a significant modulatory effect on the rates of RNA synthesis. The observed properties of NusG were consistent with a unified model where the function of this accessory factor is to promote transcriptionally downstream motion of the enzyme along the DNA template, which has the effect of forward-biasing RNAP from the pre-translocated state toward the post-translocated state.
The trigger loop (TL) is a polymorphous component of RNA polymerase (RNAP) that makes direct substrate contacts and promotes nucleotide addition when folded into an alpha-helical hairpin (trigger helices, TH). However, the roles of the TL/TH in transcript cleavage, catalysis, substrate selectivity and pausing remain ill defined. Based on in vitro assays of Escherichia coli RNAP bearing specific TL/TH alterations, we report that neither intrinsic nor regulator-assisted transcript cleavage of backtracked RNA requires formation of the TH. We find that the principal contribution of TH formation to rapid nucleotidyl transfer is steric alignment of the reactants rather than acid-base catalysis, and that the TL/TH cannot be the sole contributor to substrate selectivity. The similar effects of TL/TH substitutions on pausing and nucleotide addition provide additional support for the view that TH formation is rate-limiting for escape from nonbacktracked pauses.
The transcription termination factor Rho is a global regulator of RNA polymerase (RNAP). Although individual Rho-dependent terminators have been studied extensively, less is known about the sites of RNAP regulation by Rho on a genome-wide scale. Using chromatin immunoprecipitation and microarrays (ChIP-chip), we examined changes in the distribution of Escherichia coli RNAP in response to the Rho-specific inhibitor bicyclomycin (BCM). We found approximately 200 Rho-terminated loci that were divided evenly into 2 classes: intergenic (at the ends of genes) and intragenic (within genes). The intergenic class contained noncoding RNAs such as small RNAs (sRNAs) and transfer RNAs (tRNAs), establishing a previously unappreciated role of Rho in termination of stable RNA synthesis. The intragenic class of terminators included a previously uncharacterized set of short antisense transcripts, as judged by a shift in the distribution of RNAP in BCM-treated cells that was opposite to the direction of the corresponding gene. These Rho-terminated antisense transcripts point to a role of noncoding transcription in E. coli gene regulation that may resemble the ubiquitous noncoding transcription recently found to play myriad roles in eukaryotic gene regulation.
NusG is a conserved regulatory protein that interacts with elongation complexes (ECs) of RNA polymerase, DNA, and RNA to modulate transcription in multiple and sometimes opposite ways. In Escherichia coli, NusG suppresses pausing and increases elongation rate, enhances termination by E. coli rho and phage HK022 Nun protein, and promotes antitermination by lambdaN and in ribosomal RNA operons. We report NMR studies that suggest that E. coli NusG consists of two largely independent N- and C-terminal structural domains, NTD and CTD, respectively. Based on tests of the functions of the NTD and CTD and variants of NusG in vivo and in vitro, we find that NTD alone is sufficient to suppress pausing and enhance transcript elongation in vitro. However, neither domain alone can enhance rho-dependent termination or support antitermination, indicating that interactions of both domains with ECs are required for these processes. We propose that the two domains of NusG mediate distinct interactions with ECs: the NTD interacts with RNA polymerase and the CTD interacts with rho and other regulators, providing NusG with different combinations of interactions to effect different regulatory outcomes.
No abstract available.
In plants, unorthodox multisubunit RNA polymerases (RNAPs) play key roles in small interfering RNA (siRNA) genesis and function. In a recent issue of Molecular Cell, Ream et al. (2009) established a 12-subunit composition for Arabidopsis RNAPIV and RNAPV. Subunit and sequence divergence between RNAPIV-V and RNAPI-III suggests significant functional deviation of these intriguing RNAPs.
The trafficking patterns of the bacterial regulators of transcript elongation sigma(70), rho, NusA, and NusG on genes in vivo and the explanation for promoter-proximal peaks of RNA polymerase (RNAP) are unknown. Genome-wide, E. coli ChIP-chip revealed distinct association patterns of regulators as RNAP transcribes away from promoters (rho first, then NusA, then NusG). However, the interactions of elongating complexes with these regulators did not differ significantly among most transcription units. A modest variation of NusG signal among genes reflected increased NusG interaction as transcription progresses, rather than functional specialization of elongating complexes. Promoter-proximal RNAP peaks were offset from sigma(70) peaks in the direction of transcription and co-occurred with NusA and rho peaks, suggesting that the RNAP peaks reflected elongating, rather than initiating, complexes. However, inhibition of rho did not increase RNAP levels within genes downstream from the RNAP peaks, suggesting the peaks are caused by a mechanism other than rho-dependent attenuation.
Elongation factors NusG and RfaH evolved from a common ancestor and utilize the same binding site on RNA polymerase (RNAP) to modulate transcription. However, although NusG associates with RNAP transcribing most Escherichia coli genes, RfaH regulates just a few operons containing ops, a DNA sequence that mediates RfaH recruitment. Here, we describe the mechanism by which this specificity is maintained. We observe that RfaH action is indeed restricted to those several operons that are devoid of NusG in vivo. We also show that RfaH and NusG compete for their effects on transcript elongation and termination in vitro. Our data argue that RfaH recognizes its DNA target even in the presence of NusG. Once recruited, RfaH remains stably associated with RNAP, thereby precluding NusG binding. We envision a pathway by which a specialized regulator has evolved in the background of its ubiquitous paralogue. We propose that RfaH and NusG may have opposite regulatory functions: although NusG appears to function in concert with Rho, RfaH inhibits Rho action and activates the expression of poorly translated, frequently foreign genes.
The appearance of atmospheric oxygen from photosynthetic activity led to the evolution of aerobic respiration and responses to the resulting reactive oxygen species. In Rhodobacter sphaeroides, a photosynthetic alpha-proteobacterium, a transcriptional response to the reactive oxygen species singlet oxygen ((1)O(2)) is controlled by the group IV sigma factor sigma(E) and the anti-sigma factor ChrR. In this study, we integrated various large datasets to identify genes within the (1)O(2) stress response that contain sigma(E)-dependent promoters both within R. sphaeroides and across the bacterial phylogeny. Transcript pattern clustering and a sigma(E)-binding sequence model were used to predict candidate promoters that respond to (1)O(2) stress in R. sphaeroides. These candidate promoters were experimentally validated to nine R. sphaeroides sigma(E)-dependent promoters that control the transcription of 15 (1)O(2)-activated genes. Knowledge of the R. sphaeroides response to (1)O(2) and its regulator sigma(E)-ChrR was combined with large-scale phylogenetic and sequence analyses to predict the existence of a core set of approximately eight conserved sigma(E)-dependent genes in alpha-proteobacteria and gamma-proteobacteria. The bacteria predicted to contain this conserved response to (1)O(2) include photosynthetic species, as well as free-living and symbiotic/pathogenic nonphotosynthetic species. Our analysis also predicts that the response to (1)O(2) evolved within the time frame of the accumulation of atmospheric molecular oxygen on this planet.
Transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a GC-rich RNA hairpin followed by a U-rich tract. We used single-molecule techniques to investigate the mechanism by which three representative terminators (his, t500, and tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias translocation did not affect termination efficiency (TE). However, the force-dependent kinetics of release and the force-dependent TE of a mutant imply a forward translocation mechanism for the t500 terminator. Tension on isolated U-tracts induced transcript release in a manner consistent with RNA:DNA hybrid shearing. We deduce that different mechanisms, involving hypertranslocation or shearing, operate at terminators with different U-tracts. Tension applied to RNA at terminators suggests that closure of the final 2-3 hairpin bases destabilizes the hybrid and that competing RNA structures modulate TE. We propose a quantitative, energetic model that predicts the behavior for these terminators and mutant variants.
Bacteriophages are bacterial viruses that infect bacterial cells, and they have developed ingenious mechanisms to modify the bacterial RNA polymerase. Using a rapid, specific, single-step affinity isolation procedure to purify Escherichia coli RNA polymerase from bacteriophage T4-infected cells, we have identified bacteriophage T4-dependent modifications of the host RNA polymerase. We suggest that this methodology is broadly applicable for the identification of bacteriophage-dependent alterations of the host synthesis machinery.
Transcriptional pausing by RNA polymerase is an underlying event in the regulation of transcript elongation, yet the physical changes in the transcribing complex that create the initially paused conformation remain poorly understood. We report that this nonbacktracked elemental pause results from an active-site rearrangement whose signature includes a trigger-loop conformation positioned near the RNA 3' nucleotide and a conformation of betaDloopII that allows fraying of the RNA 3' nucleotide away from the DNA template. During nucleotide addition, trigger-loop movements or folding appears to assist NTP-stimulated translocation and to be crucial for catalysis. At a pause, the trigger loop directly contributes to the paused conformation, apparently by restriction of its movement or folding, whereas a previously postulated unfolding of the bridge helix does not. This trigger-loop-centric model can explain many properties of transcriptional pausing.
The mechanism of substrate loading in multisubunit RNA polymerase is crucial for understanding the general principles of transcription yet remains hotly debated. Here we report the 3.0-A resolution structures of the Thermus thermophilus elongation complex (EC) with a non-hydrolysable substrate analogue, adenosine-5'-[(alpha,beta)-methyleno]-triphosphate (AMPcPP), and with AMPcPP plus the inhibitor streptolydigin. In the EC/AMPcPP structure, the substrate binds to the active ('insertion') site closed through refolding of the trigger loop (TL) into two alpha-helices. In contrast, the EC/AMPcPP/streptolydigin structure reveals an inactive ('preinsertion') substrate configuration stabilized by streptolydigin-induced displacement of the TL. Our structural and biochemical data suggest that refolding of the TL is vital for catalysis and have three main implications. First, despite differences in the details, the two-step preinsertion/insertion mechanism of substrate loading may be universal for all RNA polymerases. Second, freezing of the preinsertion state is an attractive target for the design of novel antibiotics. Last, the TL emerges as a prominent target whose refolding can be modulated by regulatory factors.
We have identified minimal nucleic acid scaffolds capable of reconstituting hairpin-stabilized paused transcription complexes when incubated with RNAP either directly or in a limited step reconstitution assay. Direct reconstitution was achieved using a 29-nucleotide (nt) RNA whose 3'-proximal 9-10 nt pair to template DNA within an 11-nt noncomplementary bubble of a 39-bp duplex DNA; the 5'-proximal 18 nt of RNA forms the his pause RNA hairpin. Limited-step reconstitution was achieved on the same DNAs using a 27-nt RNA that can be 3'-labeled during reconstitution and then extended 2 nt past the pause site to assay transcriptional pausing. Paused complexes formed by either method recapitulated key features of a promoter-initiated, hairpin-stabilized paused complex, including a slow rate of pause escape, resistance to transcript cleavage and pyrophosphorolysis, and enhancement of pausing by the elongation factor NusA. These findings establish that RNA upstream from the pause hairpin and pyrophosphate are not essential for pausing and for NusA action. Reconstitution of the his paused transcription complex provides a valuable tool for future studies of protein-nucleic interactions involved in transcriptional pausing.
The architecture of cellular RNA polymerases (RNAPs) dictates that transcription can begin only after promoter DNA bends into a deep channel and the start site nucleotide (+1) binds in the active site located on the channel floor. Formation of this transcriptionally competent "open" complex (RP(o)) by Escherichia coli RNAP at the lambdaP(R) promoter is greatly accelerated by DNA upstream of base pair -47 (with respect to +1). Here we report real-time hydroxyl radical (*OH) and potassium permanganate (KMnO4) footprints obtained under conditions selected for optimal characterization of the first kinetically significant intermediate (I(1)) in RP(o) formation. .OH footprints reveal that the DNA backbone from -71 to -81 is engulfed by RNAP in I(1) but not in RP(o); downstream protection extends to approximately +20 in both complexes. KMnO4 footprinting detects solvent-accessible thymine bases in RP(o), but not in I(1). We conclude that upstream DNA wraps more extensively on RNAP in I(1) than in RP(o) and that downstream DNA (-11 to +20) occupies the active-site channel in I(1) but is not yet melted. Mapping of the footprinting data onto available x-ray structures provides a detailed model of a kinetic intermediate in bacterial transcription initiation and suggests how transient contacts with upstream DNA in I(1) might rearrange the channel to favor entry of downstream duplex DNA.
The 2006 Nobel Prize in Chemistry has been awarded to Roger Kornberg for elucidating the molecular basis of eukaryotic transcription. The prize caps a decades-long quest to unlock one of the central mysteries of molecular biology-how RNA transcripts are assembled.
The multisubunit RNAPs (RNA polymerases) found in all cellular life forms are remarkably conserved in fundamental structure, in mechanism and in their susceptibility to sequence-dependent pausing during transcription of DNA in the absence of elongation regulators. Recent studies of both prokaryotic and eukaryotic transcription have yielded an increasing appreciation of the extent to which gene regulation is accomplished during the elongation phase of transcription. Transcriptional pausing is a fundamental enzymatic mechanism that underlies many of these regulatory schemes. In some cases, pausing functions by halting RNAP for times or at positions required for regulatory interactions. In other cases, pauses function by making RNAP susceptible to premature termination of transcription unless the enzyme is modified by elongation regulators that programme efficient gene expression. Pausing appears to occur by a two-tiered mechanism in which an initial rearrangement of the enzyme's active site interrupts active elongation and puts RNAP in an elemental pause state from which additional rearrangements or regulator interactions can create long-lived pauses. Recent findings from biochemical and single-molecule transcription experiments, coupled with the invaluable availability of RNAP crystal structures, have produced attractive hypotheses to explain the fundamental mechanism of pausing.
The recently described crystal structures of multi-subunit RNA polymerases (RNAPs) reveal a conserved loop-like feature called the lid. The lid projects from the clamp domain and contacts the flap, thereby enclosing the RNA transcript in RNAP's RNA-exit channel and forming the junction between the exit channel and the main channel, which holds the RNA:DNA hybrid. In the initiating form of bacterial RNAP (holoenzyme containing sigma), the lid interacts with sigma region 3 and encloses an extended linker between sigma region 3 and sigma region 4 in place of the RNA in the exit channel. During initiation, the lid may be important for holding open the transcription bubble and may help displace the RNA from the template DNA strand. To test these ideas, we constructed and characterized a mutant RNAP from which the lid element was deleted. Deltalid RNAP exhibited dramatically reduced activity during initiation from -35-dependent and -35-independent promoters, verifying that the lid is important for stabilizing the open promoter complex during initiation. However, transcript elongation, RNA displacement, and, surprisingly, transcriptional termination all occurred normally in Deltalid RNAP. Importantly, Deltalid RNAP behaved differently from wild-type RNAP when transcribing single-stranded DNA templates where it synthesized long, persistent RNA:DNA hybrids, in contrast to efficient transcriptional arrest by wild-type RNAP.
Transcriptional elongation and termination by RNA polymerase (RNAP) are controlled by interactions among the nascent RNA, DNA, and RNAP that comprise the ternary transcription elongation complex (TEC). To probe the effects of cotranscriptionally folded RNA hairpins on elongation as well as the stability of the TEC, we developed a single-molecule assay to monitor RNA elongation by Escherichia coli RNAP molecules while applying controlled loads to the nascent RNA that favor forward translocation. Remarkably, forces up to 30 pN, twice those required to disrupt RNA secondary structure, did not significantly affect enzyme processivity, transcription elongation rates, pause frequencies, or pause lifetimes. These results indicate that ubiquitous transcriptional pausing is not a consequence of the formation of hairpins in the nascent RNA. The ability of the TEC to sustain large loads on the transcript reflects a tight binding of RNA within the TEC and has important implications for models of transcriptional termination.
Transcriptional pausing by RNA polymerase (RNAP) plays an important role in the regulation of gene expression. Defined, sequence-specific pause sites have been identified biochemically. Single-molecule studies have also shown that bacterial RNAP pauses frequently during transcriptional elongation, but the relationship of these "ubiquitous" pauses to the underlying DNA sequence has been uncertain. We employed an ultrastable optical-trapping assay to follow the motion of individual molecules of RNAP transcribing templates engineered with repeated sequences carrying imbedded, sequence-specific pause sites of known regulatory function. Both the known and ubiquitous pauses appeared at reproducible locations, identified with base-pair accuracy. Ubiquitous pauses were associated with DNA sequences that show similarities to regulatory pause sequences. Data obtained for the lifetimes and efficiencies of pauses support a model where the transition to pausing branches off of the normal elongation pathway and is mediated by a common elemental state, which corresponds to the ubiquitous pause.
Bacterial RNA polymerase (RNAP) is a complex molecular machine in which the network of interacting parts and their movements, including contacts to nascent RNA and the DNA template, are at best partially understood. The jaw domain is a part of RNAP that makes a key contact to duplex DNA as it enters the enzyme from downstream and also contacts two other parts of RNAP, the trigger loop, which lies in the RNAP secondary channel, and a sequence insertion in the Escherichia coli RNAP trigger loop that forms an external domain and also contacts downstream DNA. Deletion of the jaw domain causes defects in transcriptional pausing and in bacterial growth. We report here that these defects can be partially corrected by a limited set of substitutions in a distant part of RNAP, the product RNA-binding pocket. The product RNA-binding pocket binds nascent RNA upstream of the active site and is the binding site for the RNAP inhibitor rifampicin when RNA is absent. These substitutions have little effect on transcript elongation between pause sites and actually exacerbate jaw-deletion defects in transcription initiation, suggesting that the pausing defects may be principally responsible for the in vivo phenotype of the jaw deletion. We suggest that the counteracting effects on pausing of the alterations in the jaw and the product RNA binding site may be mediated either by effects on translocation or via allosteric communication to the RNAP active site.
The recent elucidation of crystal structures for multi-subunit RNA polymerases immediately revealed a mystery: how do nucleotide triphosphate (NTP) substrates reach an active site that is buried deep within the enzyme? The prevailing view is that NTPs enter through an approximately 20A-long secondary channel between the active site and the enzyme surface. Recently, an alternative view has been advocated; namely, NTPs enter the active site pre-bound to the DNA template from the downstream DNA portion of the main channel of the enzyme.
In bacteria, a fundamental level of gene regulation occurs by competitive association of promoter-specificity factors called sigmas with RNA polymerase (RNAP). This sigma cycle paradigm underpins much of our understanding of all transcriptional regulation. Here, we review recent challenges to the sigma cycle paradigm in the context of its essential features and of the structural basis of sigma interactions with RNAP and elongation complexes. Although sigmas can play dual roles as both initiation and elongation regulators, we suggest that the key postulate of the sigma cycle, that sigmas compete for binding to RNAP after each round of RNA synthesis, remains the central mechanism for programming transcription initiation in bacteria.
During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7 +/- 0.6 A, a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force-velocity relationship for transcription at both saturating and sub-saturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site.
Formation of productive transcription complexes after promoter escape by RNA polymerase II is a major event in eukaryotic gene regulation. Both negative and positive factors control this step. The principal negative elongation factor (NELF) contains four polypeptides and requires for activity the two-polypeptide 5,6-dichloro-1-beta-D-ribobenzimidazole-sensitivity inducing factor (DSIF). DSIF/NELF inhibits early transcript elongation until it is counteracted by the positive elongation factor P-TEFb. We report a previously undescribed activity of DSIF/NELF, namely inhibition of the transcript cleavage factor TFIIS. These two activities of DSIF/NELF appear to be mechanistically distinct. Inhibition of nucleotide addition requires > or = 18 nt of nascent RNA, whereas inhibition of TFIIS occurs at all transcript lengths. Because TFIIS promotes escape from promoter-proximal pauses by stimulating cleavage of back-tracked nascent RNA, TFIIS inhibition may help DSIF/NELF negatively regulate productive transcription.
The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the beta' subunit. The DNA was amplified and hybridized to "tiled" oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known sigma70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique.
Bacterial RNA polymerase is a common target for many antibiotics. In two recent papers in Cell and Molecular Cell, and describe a structural basis for inhibition of bacterial RNA polymerase by the antibiotic streptolydigin. Streptolydigin may prevent distortion of a "bridge" alpha helix postulated to occur during the nucleotide addition cycle of RNA polymerase or may block a small movement of the bridge helix that helps load nucleotide triphosphates into the active site.
No abstract available.
The Escherichia coli RNA polymerase beta subunit contains a flexible flap domain that interacts with region 4 of sigma(70) to position it for recognition of the -35 element of promoters. We report that this function depends on a hydrophobic patch on one face of the short stretch of alpha helix located at the tip of the flap domain, called the flap-tip helix. Disruption of the hydrophobic patch by the substitution of hydrophilic or charged amino acids resulted in a loss of the interaction between the flap and sigma region 4, as determined by protease sensitivity assays, and impaired transcription from -35-dependent promoters. We suggest that contact of the flap-tip helix hydrophobic patch to the sigma region 4 hydrophobic core is essential for stable interaction of the flap-tip helix with region 4. This contact allowed region 4.2 recognition of the -35 promoter element and appeared to stabilize region 4 interaction with the beta' Zn(2+) binding domain. Our studies failed to detect any role for sigma region 1.1 in establishing or maintaining the flap-sigma region 4 interaction, consistent with recent reports placing sigma region 1.1 in the downstream DNA channel.
Transcriptional pausing by human RNA polymerase II (RNAPII) in the HIV-1 LTR is caused principally by a weak RNA:DNA hybrid that allows rearrangement of reactive or catalytic groups in the enzyme's active site. This rearrangement creates a transiently paused state called the unactivated intermediate that can backtrack into a more long-lived paused species. We report that three different regions of the not-yet-transcribed DNA just downstream of the pause site affect the duration of the HIV-1 pause, and also can influence pause formation. Downstream DNA in at least one region, a T-tract from +5 to +8, increases pause duration by specifically affecting the unactivated intermediate, without corresponding effects on the active or backtracked states. We suggest this effect depends on RNAPII-modulated DNA plasticity and speculate it is mediated by the "trigger loop" thought to participate in RNAP's catalytic cycle. These findings provide a new framework for understanding downstream DNA effects on RNAP.
New crystal structures of transcription complexes formed by bacteriophage T7 RNA polymerase reveal a nucleotide-addition cycle driven by active-site conformational changes similar to those observed in DNA polymerases, and suggest provocative hypotheses for the more complex multisubunit RNA polymerases of free-living organisms.
Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogeneous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a approximately 2650-bp DNA during transcript elongation in vitro at 0.5 mm nucleoside triphosphates. Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy. The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription. With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian. However, the width of the Gaussian, sigma = 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s). The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers. These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution.
Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo. Its low error rate may be achieved by means of a 'proofreading' mechanism comprised of two sequential events. The first event (backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 3' end of the nascent RNA transcript away from the enzyme active site. The second event (endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site. Here, by combining ultrastable optical trapping apparatus with a novel two-bead assay to monitor transcriptional elongation with near-base-pair precision, we observed backtracking and recovery by single molecules of RNAP. Backtracking events ( approximately 5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to >30 min. Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.
Bacterial RNA polymerase (RNAP) responds to formation of RNA secondary structures (hairpins) in newly synthesized RNA. Depending on the spacing of the hairpin from the RNA 3' end and the intervening RNA sequence, the hairpin can prolong pausing or cause transcriptional termination. At the his pause site, the pause hairpin contacts a flexible domain on RNAP called the flap, which forms a critical part of a hairpin-interaction site on the enzyme. We report that pause hairpin-flap interaction stabilizes an inhibited configuration of RNAP's active site without changing RNAP's translocation register. The distal part of the flap (the flap tip) is required for the hairpin to affect the active site, but not for hairpin formation. In contrast, the flap tip is not required for intrinsic termination, but can modulate it at suboptimal termination signals.
Bacterial sigma factors compete for binding to RNA polymerase (RNAP) to control promoter selection, and in some cases interact with RNAP to regulate at least the early stages of transcript elongation. However, the effective concentration of sigmas in vivo, and the extent to which sigma can regulate transcript elongation generally, are unknown. We report that tethering sigma70 to all RNAP molecules via genetic fusion of rpoD to rpoC (encoding sigma70 and RNAP's beta' subunit, respectively) yields viable Escherichia coli strains in which alternative sigma-factor function is not impaired. beta'::sigma70 RNAP transcribed DNA normally in vitro, but allowed sigma70-dependent pausing at extended -10-like sequences anywhere in a transcriptional unit. Based on measurement of the effective concentration of tethered sigma70, we conclude that the effective concentration of sigma70 in E. coli (i.e., its thermodynamic activity) is close to its bulk concentration. At this level, sigma70 would be a bona fide elongation factor able to direct transcriptional pausing even after its release from RNAP during promoter escape.
RNA polymerase (RNAP) transcribes DNA discontinuously, with periods of rapid nucleotide addition punctuated by frequent pauses. We investigated the mechanism of transcription by measuring the effect of both hindering and assisting forces on the translocation of single Escherichia coli transcription elongation complexes, using an optical trapping apparatus that allows for the detection of pauses as short as one second. We found that the vast majority of pauses are brief (1-6 s at 21 degrees C, 1 mM NTPs), and that the probability of pausing at any particular position on a DNA template is low and fairly constant. Neither the probability nor the duration of these ubiquitous pauses was affected by hindering or assisting loads, establishing that they do not result from the backtracking of RNAP along the DNA template. We propose instead that they are caused by a structural rearrangement within the enzyme.
RNA polymerase (RNAP) is the central enzyme of gene expression. Despite availability of crystal structures, details of its nucleotide addition cycle remain obscure. We describe bacterial RNAP inhibitors (the CBR703 series) whose properties illuminate this mechanism. These compounds inhibit known catalytic activities of RNAP (nucleotide addition, pyrophosphorolysis, and Gre-stimulated transcript cleavage) but not translocation of RNA or DNA when translocation is uncoupled from catalysis. CBR703-resistance substitutions occur on an outside surface of RNAP opposite its internal active site. We propose that CBR703 compounds inhibit nucleotide addition allosterically by hindering movements of active site structures that are linked to the CBR703 binding site through a bridge helix.
Microcin J25 (MccJ25) is a 21-amino acid peptide inhibitor active against the DNA-dependent RNA polymerase of Gram negative bacteria. Previously, the structure of MccJ25 was reported to be a head-to-tail circle, cyclo(-G(1)GAGHVPEYF(10)VGIGTPISFY(20)G-). On the basis of biochemical studies, mass spectrometry, and NMR, we show that this structure is incorrect, and that the peptide has an extraordinary structural fold. MccJ25 contains an internal lactam linkage between the alpha-amino group of Gly1 and the gamma-carboxyl of Glu8. The tail (Tyr9-Gly21) passes through the ring (Gly1-Glu8), with Phe19 and Tyr20 straddling each side of the ring, sterically trapping the tail in a noncovalent interaction we call a lassoed tail.
Bacteriophage lambda Q-protein stably binds and modifies RNA polymerase (RNAP) to a termination-resistant form. We describe amino acid substitutions in RNAP that disrupt Q-mediated antitermination in vivo and in vitro. The positions of these substitutions in the modeled RNAP/DNA/RNA ternary elongation complex, and their biochemical properties, suggest that they do not define a binding site for Q in RNAP, but instead act by impairing interactions among core RNAP subunits and nucleic acids that are essential for Q modification. A specific conjecture is that Q modification stabilizes interactions of RNAP with the DNA/RNA hybrid and optimizes alignment of the nucleic acids in the catalytic site. Such changes would inhibit the activity of the RNA hairpin of an intrinsic terminator to disrupt the 5'-terminal bases of the hybrid and remove the RNA 3' terminus from the active site.
The study of mutant enzymes can reveal important details about the fundamental mechanism and regulation of RNA polymerase, the central enzyme of gene expression. However, such studies are complicated by the multisubunit structure of RNA polymerase and by its indispensability for cell growth. Previously, mutant RNA polymerases have been produced by in vitro assembly from isolated subunits or by in vivo assembly upon overexpression of a single mutant subunit. Both approaches can fail if the mutant subunit is toxic or incorrectly folded. Here we describe an alternative strategy, co-overexpression and in vivo assembly of RNA polymerase subunits, and apply this method to characterize the role of sequence insertions present in the Escherichia coli enzyme. We find that co-overexpression of its subunits allows assembly of an RNA polymerase lacking a 188-amino acid insertion in the beta' subunit. Based on experiments with this and other mutant E. coli enzymes with precisely excised sequence insertions, we report that the beta' sequence insertion and, to a lesser extent, an N-terminal beta sequence insertion confer characteristic stability to the open initiation complex, frequency of abortive initiation, and pausing during transcript elongation relative to RNA polymerases, such as that from Bacillus subtilis, that lack the sequence insertions.
A mutation in the conserved segment of the rpoC gene, which codes for the largest RNA polymerase (RNAP) subunit, beta', was found to make Escherichia coli cells resistant to microcin J25 (MccJ25), a bactericidal 21-amino acid peptide active against Gram-negative bacteria (Delgado, M. A., Rintoul, M. R., Farias, R. N., and Salomon, R. A. (2001) J. Bacteriol. 183, 4543-4550). Here, we report that mutant RNAP prepared from MccJ25-resistant cells, but not the wild-type RNAP, is resistant to MccJ25 in vitro, thus establishing that RNAP is a true cellular target of MccJ25. We also report the isolation of additional rpoC mutations that lead to MccJ25 resistance in vivo and in vitro. The new mutations affect beta' amino acids in evolutionarily conserved segments G, G', and F and are exposed into the RNAP secondary channel, a narrow opening that connects the enzyme surface with the catalytic center. We also report that previously known rpoB (RNAP beta subunit) mutations that lead to streptolydigin resistance cause resistance to MccJ25. We hypothesize that MccJ25 inhibits transcription by binding in RNAP secondary channel and blocking substrate access to the catalytic center.
Regulation of RNA polymerase during initiation, elongation, and termination of transcription is mediated in part by interactions with intrinsic regulatory signals encoded in the RNA and DNA that contact the enzyme. These interactions include contacts to an 8-9-bp RNA:DNA hybrid within the active-site cleft of the enzyme, contacts to the melted nontemplate DNA strand in the vicinity of the hybrid, contacts to exiting RNA upstream of the hybrid, and contacts to approximately 20 bp of duplex DNA downstream of the active site. Based on characterization of an amino acid substitution (G1161R) and a deletion (Delta1149-1190) in the jaw domain of the bacterial RNA polymerase largest subunit (beta'), we report here that contacts of the jaw domain to downstream DNA at the leading edge of the transcription complex contribute to regulation during all three phases of transcription. The results provide insight into the role of the jaw domain-downstream DNA contact in transcriptional initiation and pausing and suggest possible explanations for the previously reported isolation of the jaw mutants based on reduced ColEI plasmid replication.
The transcriptional regulatory protein RfaH controls expression of several operons that encode extracytoplasmic components in bacteria. Regulation by RfaH occurs during transcript elongation and depends on a 5'-proximal, transcribed nucleic acid sequence called ops that induces transcriptional pausing in vitro and in vivo. We report that RfaH recognizes RNA polymerase transcribing RfaH-regulated operons by interacting with the ops sequence in the exposed nontemplate DNA strand of ops-paused transcription complexes. Although RfaH delays escape from the ops pause, once escape occurs, RfaH enhances elongation by suppressing pausing and rho-dependent termination without apparent involvement of other accessory proteins. This activity predicts a cumulative antitermination model for RfaH's regulation of ops-containing operons in vivo.
Human RNA polymerase II recognizes a strong transcriptional pause signal in the initially transcribed region of HIV-1. We report the use of a limited-step transcription assay to dissect the mechanism underlying recognition of and escape from this HIV-1 pause. Our results suggest that the primary determinant of transcriptional pausing is a relatively weak RNA:DNA hybrid that triggers backtracking of RNA polymerase II along the RNA and DNA chains and displaces the RNA 3' OH from the active site. In contrast, two alternative RNA secondary structures, TAR and anti-TAR, are not required for pausing and affect it only indirectly, rather than through direct interaction with RNA polymerase II. TAR accelerates escape from the pause, but anti-TAR inhibits formation of TAR prior to pause escape. The behavior of RNA polymerase II at a mutant pause signal supports a two-step, non-equilibrium mechanism in which the rate-determining step is a conformational change in the enzyme, rather than the changes in nucleic-acid base-pairing that accompany backtracking.
The interaction of RNA polymerase and its initiation factors is central to the process of transcription initiation. To dissect the role of this interface, we undertook the identification of the contact sites between RNA polymerase and sigma(70), the Escherichia coli initiation factor. We identified nine mutationally verified interaction sites between sigma(70) and specific domains of RNA polymerase and provide evidence that sigma(70) and RNA polymerase interact in at least a two-step process. We propose that a cycle of changes in the interface of sigma(70) with core RNA polymerase is associated with progression through the process of transcription initiation.
No abstract available.
DNA, RNA, and regulatory molecules control gene expression through interactions with RNA polymerase (RNAP). We show that a short alpha helix at the tip of the flaplike domain that covers the RNA exit channel of RNAP contacts a nascent RNA stem-loop structure (hairpin) that inhibits transcription, and that this flap-tip helix is required for activity of the regulatory protein NusA. Protein-RNA cross-linking, molecular modeling, and effects of alterations in RNAP and RNA all suggest that a tripartite interaction of RNAP, NusA, and the hairpin inhibits nucleotide addition in the active site, which is located 65 angstroms away. These findings favor an allosteric model for regulation of transcript elongation.
Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals. Some of these signals are conserved throughout evolution, whereas others are species specific. In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species, Escherichia coli and Bacillus subtilis, using a panel of in vitro transcription assays. We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals. Both enzymes responded similarly to other pause and termination signals and to the general E. coli elongation factors NusA and GreA. We also demonstrate that, although promoter recognition depends largely on the sigma subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme. We hypothesize that differences in signal recognition are due to the changes in contacts made between the beta and beta' subunits and the downstream DNA duplex.
Bacillus subtilis core RNA polymerase, containing a His(6)-fusion to the C-terminus of the beta' subunit, was isolated by Ni-NTA, Superdex 200 gel filtration, and Mono Q anion-exchange chromatography. The purified core enzyme was shown to be free of the major sigma factor(A) and the transcription factors NusA and GreA. The purification procedure can be completed within 1 working day, is scalable, and yields highly purified and active core RNA polymerase.
Transcript elongation by RNA polymerase is discontinuous and interrupted by pauses that play key regulatory roles. We show here that two different classes of pause signals punctuate elongation. Class I pauses, discovered in enteric bacteria, depend on interaction of a nascent RNA structure with RNA polymerase to displace the 3' OH away from the catalytic center. Class II pauses, which may predominate in eukaryotes, cause RNA polymerase to slide backwards along DNA and RNA and to occlude the active site with nascent RNA. These pauses differ in their responses to antisense oligonucleotides, pyrophosphate, GreA, and general elongation factors NusA and NusG. In contrast, substitutions in RNA polymerase that increase or decrease the rate of RNA synthesis affect both pause classes similarly. We propose that both pause classes, as well as arrest and termination, arise from a common intermediate that itself binds NTP substrate weakly.
Using an optical-trap/flow-control video microscopy technique, we followed transcription by single molecules of Escherichia coli RNA polymerase in real time over long template distances. These studies reveal that RNA polymerase molecules possess different intrinsic transcription rates and different propensities to pause and stop. The data also show that reversible pausing is a kinetic intermediate between normal elongation and the arrested state. The conformational metastability of RNA polymerase revealed by this single-molecule study of transcription has direct implications for the mechanisms of gene regulation in both bacteria and eukaryotes.
To identify the location of a domain of the beta-subunit of Escherichia coli RNA polymerase (RNAP) on the three-dimensional structure, we developed a method to tag a nonessential surface of the multisubunit enzyme with a protein density easily detectable by electron microscopy and image processing. Four repeats of the IgG-binding domain of Staphylococcus aureus protein A were inserted at position 998 of the E. coli RNAP beta-subunit. The mutant RNAP supported E. coli growth and showed no apparent functional defects in vitro. The structure of the mutant RNAP was determined by cryoelectron microscopy and image processing of frozen-hydrated helical crystals. Comparison of the mutant RNAP structure with the previously determined wild-type RNAP structure by Fourier difference analysis at 20-A resolution directly revealed the location of the inserted protein domain, thereby locating the region around position 998 of the beta-subunit within the RNAP three-dimensional structure and refining a model for the subunit locations within the enzyme.
Cessation of transcription at specific terminator DNA sequences is used by viruses, bacteria, and eukaryotes to regulate the expression of downstream genes, but the mechanisms of transcription termination are poorly characterized. To elucidate the kinetic mechanism of termination at the intrinsic terminators of enteric bacteria, we observed, by using single-molecule light microscopy techniques, the behavior of surface-immobilized Escherichia coli RNA polymerase (RNAP) molecules in vitro. An RNAP molecule remains at a canonical intrinsic terminator for approximately 64 s before releasing DNA, implying the formation of an elongation-incompetent (paused) intermediate by transcription complexes that terminate but not by those that read through the terminator. Analysis of pause lifetimes establishes a complete minimal mechanism of termination in which paused intermediate formation is both necessary and sufficient to induce release of RNAP at the terminator. The data suggest that intrinsic terminators function by a nonequilibrium process in which terminator effectiveness is determined by the relative rates of nucleotide addition and paused state entry by the transcription complex.
No abstract available.
Using either highly purified RNA polymerase II (pol II) elongation complexes assembled on oligo(dC)-tailed templates or promoter-initiated (extract-generated) pol II elongation complexes, the precise 3" ends of transcripts produced during transcription in vitro at several human c- and N- myc pause, arrest and termination sites were determined. Despite a low overall similarity between the entire c- and N- myc first exon sequences, many positions of pol II pausing, arrest or termination occurred within short regions of related sequence shared between the c- and N- myc templates. The c- and N- myc genes showed three general classes of sequence conservation near intrinsic pause, arrest or termination sites: (i) sites where arrest or termination occurred after the synthesis of runs of uridines (Us) preceding the transcript 3" end, (ii) sites downstream of potential RNA hairpins and (iii) sites after nucleotide addition following either a U or a C or following a combination of several pyrimidines near the transcript 3" end. The finding that regions of similarity occur near the sites of pol II pausing, arrest or termination suggests that the mechanism of c- and N- myc regulation at the level of transcript elongation may be similar and not divergent as previously proposed.
We compared in vitro transcription-initiated folding of the ribozyme from Bacillus subtilis RNase P to refolding from the full-length, denatured state by monitoring the appearance of its catalytic activity. At 37 degrees C, Mg(2+)-initiated refolding of the wild type and a circularly permutate ribozyme takes minutes and is limited by a kinetic trap. Transcription by T7 RNA polymerase alters the folding pathway of both RNAs and introduces new kinetic traps. Transcription by the core Escherichia coli RNA polymerase yields the same result, in spite of its 4-fold-slower elongation rate. However, the presence of its elongation factor NusA accelerates more than 10-fold the transcription-initiated folding of the circularly, permutated ribozyme by E. coli RNA polymerase. The effect of NusA likely is caused by its enhancement of transcriptional pausing because NusA did not accelerate transcription-initiated folding using a mutant RNA polymerase that failed to pause or respond to NusA during ribozyme synthesis. We conclude that both transcription and specific pausing therein can alter RNA-folding pathways.
No abstract available.
RNA polymerase (RNAP) moves along DNA while carrying out transcription, acting as a molecular motor. Transcriptional velocities for single molecules of Escherichia coli RNAP were measured as progressively larger forces were applied by a feedback-controlled optical trap. The shapes of RNAP force-velocity curves are distinct from those of the motor enzymes myosin or kinesin, and indicate that biochemical steps limiting transcription rates at low loads do not generate movement. Modeling the data suggests that high loads may halt RNAP by promoting a structural change which moves all or part of the enzyme backwards through a comparatively large distance, corresponding to 5 to 10 base pairs. This contrasts with previous models that assumed force acts directly upon a single-base translocation step.
Nascent RNA structures may regulate RNA chain elongation either directly through interaction with RNA polymerase or indirectly by disrupting nascent RNA contacts with polymerase or DNA. To distinguish these mechanisms we tested whether the effects of the his leader pause RNA hairpin could be mimicked by pairing of antisense DNA or RNA oligonucleotides to the nascent transcript. The his pause hairpin inhibits nucleotide addition when it forms 11 nucleotides from the transcript 3' end. It also can terminate transcription when base changes extend its stem to </=8 nucleotides from the 3' end. All oligonucleotides that disrupted the pause hairpin reduced the dwell time of RNA polymerase at the pause site dramatically, even when they mimicked the 11-nucleotide 3'-proximal RNA spacing or created a suitably positioned RNA loop. Oligonucleotides that paired </=8 nucleotides from the pause RNA 3' end could trigger transcript release, but only when added to an already paused complex. These results argue that direct interaction of a nascent RNA hairpin with RNA polymerase delays escape from a pause, but that indirect effects of a hairpin may trigger transcript release from a paused complex. Resistance of the paused complex to pyrophosphorolysis and its reversal by antisense oligonucleotides further suggest that interaction of the pause hairpin with RNA polymerase disengages the RNA 3' end from the active site.
No abstract available.
A strong transcriptional pause delays human RNA polymerase II three nt after the last potentially paired base in HIV-1 TAR, the RNA structure that binds the transactivator protein Tat. We report here that the HIV-1 pause depends in part on an alternative RNA structure (the HIV-1 pause hairpin) that competes with formation of TAR. By probing the nascent RNA structure in halted transcription complexes, we found that the transcript folds as the pause hairpin before and at the pause, and rearranges to TAR concurrent with or just after escape from the pause. The pause signal triggers a 2 nt reverse translocation by RNA polymerase that may block the active site and be counteracted by formation of TAR. Thus, the HIV-1 pause site modulates nascent RNA rearrangement from a structure that favors pausing to one that both recruits Tat and promotes escape from the pause.
No abstract available.
The rpoB and rpoC genes of eubacteria and archaea, coding, respectively, for the beta and beta'-like subunits of DNA-dependent RNA polymerase, are organized in an operon with rpoB always preceding rpoC. Here, we show that in Escherichia coli the two genes can be fused and that the resulting 2751-amino acid beta::beta' fusion polypeptide assembles into functional RNA polymerase in vivo and in vitro. The results establish that the C terminus of the beta subunit and the N terminus of the beta' subunit are in close proximity to each other on the surface of the assembled RNA polymerase during all phases of the transcription cycle and also suggest that RNA polymerase assembly in vivo may occur co-translationally.
RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all rho-independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3' end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to beta904-950 replaced crosslinking to beta' and to other parts of beta that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3' end in the paused complex and be related to events at rho-independent terminators.
Transcription is delayed in the leader regions of the Escherichia coli trp and his operons by multipartite pause signals that consist of four components: a nascent RNA structure (the pause hairpin), the 10 or 11 nt 3'-proximal region between the pause hairpin and the RNA 3' end, the bases in the active site, and approximately 14 bp of duplex DNA downstream from the pause site. Results described in the accompanying paper suggest that the his pause hairpin slows nucleotide addition via interaction with an easily disordered surface on RNA polymerase. Here we report that the four pause signal components slow nucleotide addition in a single kinetic intermediate. Formation of the paused transcription complex, in contrast, involves synergistic effects of RNA and DNA sequences that select the wild-type pause site from among several adjacent possibilities. Extending the pause hairpin with one G x C base-pair reduces pausing, apparently by interfering with pause hairpin interaction; adding a second C x G base-pair that reduces the 3'-proximal RNA to 9 nt or less (within the 7 to 9 nt characteristic of rho-independent terminators) induces transcript release. We propose that escape from the pause is governed by a rate-limiting isomerization that may require substrate NTP binding to re-establish the active site geometry, whereas transcript release and termination ensue when the hairpin interaction is weakened and isomerization to an active conformation is blocked.
We examined the effects of neutral salts and the non-ionic solute 2-methyl,-2,4-pentanediol (MPD) on transcript elongation by Escherichia coli RNA polymerase and on pausing induced by the multipartite his leader pause signal. All solutes tested slowed the overall rate of elongation, with anions showing the dominant effects in the order: (most inhibitory) HPO4(2-) > OAc- > SO4(2-) > ClO4- > I- approximately NO3- > Br approximately Cl- approximately MPD (least inhibitory). Although the protein structure-stabilizing anions HPO4(2-), OAc-, and SO4(2-) also increased the pause half-life at the his leader pause site, the remaining solutes accelerated escape from pause site in the order: (greatest acceleration) NO3- > ClO4- > I- > Br- > Cl- > MPD (least acceleration). Cl(-)-induced acceleration of escape from the pause site also occurred on mutant templates altered for the 3'-proximal region, RNA 3' end, or downstream DNA. The effect was eliminated, however, by base substitutions that destabilize the pause RNA hairpin or that extend it toward the 3' end. This "perfect hairpin" itself reduced the pause half-life by a factor of 3. We suggest that the pause RNA hairpin stabilizes a paused conformation of the transcription complex through an interaction with an easily disordered region of RNA polymerase. Extending the stem of the pause hairpin may disrupt the interaction by altering the position of the hairpin in the transcription complex. Anions may either compete for the interaction directly or disorder the site of hairpin interaction by chaotropic effects. We suggest that the negative effect of structure-stabilizing anions like OAc- and SO4(2-) may reflect passage of RNA polymerase through significantly different conformations during rapid elongation, some of which may expose hydrophobic surface.
No abstract available.
Force-extension (F-x) relationships were measured for single molecules of DNA under a variety of buffer conditions, using an optical trapping interferometer modified to incorporate feedback control. One end of a single DNA molecule was fixed to a coverglass surface by means of a stalled RNA polymerase complex. The other end was linked to a microscopic bead, which was captured and held in an optical trap. The DNA was subsequently stretched by moving the coverglass with respect to the trap using a piezo-driven stage, while the position of the bead was recorded at nanometer-scale resolution. An electronic feedback circuit was activated to prevent bead movement beyond a preset clamping point by modulating the light intensity, altering the trap stiffness dynamically. This arrangement permits rapid determination of the F-x relationship for individual DNA molecules as short as -1 micron with unprecedented accuracy, subjected to both low (approximately 0.1 pN) and high (approximately 50 pN) loads: complete data sets are acquired in under a minute. Experimental F-x relationships were fit over much of their range by entropic elasticity theories based on worm-like chain models. Fits yielded a persistence length, Lp, of approximately 47 nm in a buffer containing 10 mM Na1. Multivalent cations, such as Mg2+ or spermidine 3+, reduced Lp to approximately 40 nm. Although multivalent ions shield most of the negative charges on the DNA backbone, they did not further reduce Lp significantly, suggesting that the intrinsic persistence length remains close to 40 nm. An elasticity theory incorporating both enthalpic and entropic contributions to stiffness fit the experimental results extremely well throughout the full range of extensions and returned an elastic modulus of approximately 1100 pN.
We tested the susceptibility of nucleic acid strands in a halted transcription elongation complex to digestion by micrococcal nuclease (MN). The 16-nucleotide nascent RNA was protected within RNA polymerase. A 27-28-nucleotide template strand DNA fragment also was resistant to MN digestion. However, the upstream half of the nontemplate DNA within this region was digested rapidly by MN, suggesting that the nontemplate strand emerges from the RNA polymerase near the middle of the melted transcription bubble with the bases oriented away from the enzyme surface. MN cleavage of the exposed nontemplate DNA shifted polymerase backward, making it unable to extend the RNA chain. However, the MN-trimmed G16 complexes could be reactivated by GreB-stimulated cleavage of the nascent RNA. These results favor a model of transcriptional arrest involving upstream slippage of RNA polymerase along the RNA and DNA chains. They also suggest that the exposed segment of nontemplate DNA may directly or indirectly stabilize the lateral position of the transcription complex along the DNA.
No abstract available.
The beta subunit of prokaryotic RNA polymerase shares significant sequence similarity with its eukaryotic and archaeal counterparts across most of the protein. Nine segments of particularly high similarity have been identified and are termed segments A through I. We have isolated severely defective Escherichia coli RNA polymerase mutants, most of which are unable to support bacterial growth. The majority of the substitutions affect residues in one of the conserved segments of beta, including invariant residues in segments D (amino acids 548 to 577), E (amino acids 660 to 678), and I (amino acids 1198 to 1296). In addition, recessive-lethal mutations that affect residues highly conserved only among prokaryotes were identified. They include a substitution in the extreme amino terminus of beta, a region in which no substitutions have previously been identified, and one rpoB mutation that truncates the polypeptide without abolishing minimal polymerase function in vitro. To examine the recessive-lethal alleles in vitro, we devised a novel method to remove nonmutant enzyme from RNA polymerase preparations by affinity tagging the chromosomal rpoB gene. In vitro examination of a subset of purified recessive-lethal RNA polymerases revealed that several substitutions, including all of those altering conserved residues in segment I, severely decrease transcript elongation and increase termination. We discuss the insights these mutants lend to a structure-function analysis of RNA polymerase.
No abstract available.
Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all rho-dependent and rho-independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (betaN1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (beta'R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for betaN1072H. In contrast, beta'R352C and two adjacent substitutions (beta'G351S and beta'S350F), but not betaN1072H, formed open complexes of greatly reduced stability. The sequence in this region of beta' modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in beta', however, since the Rifr mutation rpoB2 in beta also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of beta region H in nucleoside triphosphate-binding and nucleotide addition and of beta' region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.
The force produced by a single molecule of Escherichia coli RNA polymerase during transcription was measured optically. Polymerase immobilized on a surface was used to transcribe a DNA template attached to a polystyrene bead 0.5 micrometer in diameter. The bead position was measured by interferometry while a force opposing translocation of the polymerase along the DNA was applied with an optical trap. At saturating nucleoside triphosphate concentrations, polymerase molecules stalled reversibly at a mean applied force estimated to be 14 piconewtons. This force is substantially larger than those measured for the cytoskeletal motors kinesin and myosin and exceeds mechanical loads that are estimated to oppose transcriptional elongation in vivo. The data are consistent with efficient conversion of the free energy liberated by RNA synthesis into mechanical work.
Mutations conferring streptolydigin resistance onto Escherichia coli RNA polymerase have been found exclusively in the beta subunit (Heisler, L. M., Suzuki, H., Landick, R., and Gross, C. A. (1993) J. Biol. Chem. 268, 25369-25375). We report here the isolation of a streptolydigin-resistant mutation in the E. coli rpoC gene, encoding the beta' subunit. The mutation is the Phe793-->Ser substitution, which occurred in an evolutionarily conserved segment of the beta' subunit. The homologous segment in the eukaryotic RNA polymerase II largest subunit harbors mutations conferring alpha-amanitin resistance. Both streptolydigin and alpha-amanitin are inhibitors of transcription elongation. Thus, the two antibiotics may inhibit transcription in their respective systems by a similar mechanism, despite their very different chemical nature.
The Escherichia coli rpoB gene, which codes for the 1342-residue beta subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To test whether these regions demarcate domains of the RNAP beta subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these beta-subunit polypeptide fragments, when added with purified recombinant beta', sigma, and alpha subunits of RNAP, reconstitute a functional enzyme in vitro. These results demonstrate that the beta subunit is composed of at least three distinct domains and open another avenue for in vitro studies of RNAP assembly and structure.
A central enigma of transcriptional regulation is how the normally efficient transcription elongation complex stops at pause and termination signals. One possibility, raised by the discovery that RNA polymerase sometimes contracts its DNA footprint, is that discontinuous movements contribute to recognizing these signals. We report that E. coli RNA polymerase responds to sequences immediately downstream and upstream from the his leader pause site by changing neither its downstream DNA contact nor its upstream RNA contact for 8 bp preceding the pause. This compressed complex isomerizes to a paused conformation by an approximately 10 bp jump of its downstream DNA contact and simultaneous extrusion of an RNA hairpin that stabilizes the paused conformation. We suggest pausing and termination could be alternative outcomes of a similar isomerization that depend on the strength of contacts to 3'-proximal RNA remaining after the jump.
No abstract available.
Schafer et al. (Nature 352:444-448 (1991)) devised the tethered particle motion (TPM) method to detect directly the movement of single, isolated molecules of a processive nucleic acid polymerase along a template DNA molecule. In TPM studies, the polymerase molecule is immobilized on a glass surface, and a particle (e.g., a 0.23 microns diameter polystyrene bead) is attached to one end of the enzyme-bound DNA molecule. Time-resolved measurements of the DNA contour length between the particle and the immobilized enzyme (the "tether length") are made by determining the magnitude of the Brownian motion of the DNA-tethered particle using light microscopy and digital image processing. We report here improved sample preparation methods that permit TPM data collection on transcript elongation by the Escherichia coli RNA polymerase at rates (approximately 10(2)-fold higher than those previously obtained) sufficient for practical use of microscopic kinetics techniques to analyze polymerase reaction mechanisms. In earlier TPM experiments, calculation of tether length from the observed Brownian motion was based on an untested numerical simulation of tethered bead Brownian motion. Using the improved methods, we have now empirically validated the TPM technique for tether lengths of 308-1915 base pairs (bp) using calibration specimens containing particles tethered by individual DNA molecules of known lengths. TPM analysis of such specimens yielded a linear calibration curve relating observed Brownian motion to tether length and allowed determination of the accuracy of the technique and measurement of how temporal bandwidth, tether length, and other experimental variables affect measurement precision. Under a standard set of experimental conditions (0.23 microns diameter bead, 0.23 Hz bandwidth, 23 degrees), accuracy is 108 and 258 bp r.m.s. at tether lengths of 308 and 1915 bp, respectively. Precision improves linearly with decreasing tether length to an extrapolated instrumentation limit of 10 bp r.m.s. and improves proportionally to the inverse square root of measurement bandwidth (1.9 x 10(2) bp Hz-1/2 for 1090-bp tethers). Measurements on large numbers of individual polymerase molecules reveal that time-averaged single-molecule elongation rates are more variable than is predicted from the random error in TPM measurements, demonstrating that the surface-immobilized RNA polymerase molecules are kinetically heterogeneous.
To identify regions of the largest subunit of RNA polymerase that are potentially involved in transcript elongation and termination, we have characterized amino acid substitutions in the beta' subunit of Escherichia coli RNA polymerase that alter expression of reporter genes preceded by terminators in vivo. Termination-altering substitutions occurred in discrete segments of beta', designated 2, 3a, 3b, 4a, 4b, 4c, and 5, many of which are highly conserved in eukaryotic homologs of beta'. Region 2 substitutions (residues 311-386) are tightly clustered around a short sequence that is similar to a portion of the DNA-binding cleft in E. coli DNA polymerase I. Region 3b (residues 718-798) corresponds to the segment of the largest subunit of RNA polymerase II in which amanitin-resistance substitutions occur. Region 4a substitutions (residues 933-936) occur in a segment thought to contact the transcript 3' end. Region 5 substitutions (residues 1308-1356) are tightly clustered in conserved region H near the carboxyl terminus of beta'. A representative set of mutant RNA polymerases were purified and revealed unexpected variation in percent termination at six different rho-independent terminators. Based on the location and properties of these substitutions, we suggest a hypothesis for the relationship of subunits in the transcription complex.
GreA is a 17.6 kDa protein from Escherichia coli that induces cleavage of the nascent transcript in the elongating complex of RNA polymerase, followed by release of the 3'-terminal fragment. Crystals of GreA have been obtained from polyethylene glycol 4000, 2-propanol and sodium citrate, pH 5.6 and have been propagated by a novel seeding procedure. The crystals diffract beyond 2 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 101.7 A, b = 42.22 A, c = 40.05 A and with one molecule in the asymmetric unit.
GreA- and GreB-induced transcript cleavage drives reverse translocation of Escherichia coli RNA polymerase on a DNA template in the absence of NTPs (Feng, G.-H., Lee, D. N., Wang, D., Chan, C. L., and Landick, R. (1994) J. Biol. Chem. 269, 22282-22294, accompanying report). During transcript elongation, the sizes of the DNA footprint and the single-stranded transcription bubble vary markedly among transcription complexes halted at different template positions. To test whether transcription complex intermediates formed during transcript cleavage-induced reverse translocation also display heterogeneous conformations at different template positions, we examined the structures of two different transcription complexes before and after GreA treatment. Transcription complexes halted at position +16 after initiation at the T7 A1 promoter or paused at the trpL pause site exhibited strong blocks to transcript cleavage after removal of 6 to 10 nucleotides. In both cases, the down-stream contact between RNA polymerase and DNA moved little during transcript cleavage, thereby increasing its distance from the active site, whereas the upstream DNA contact and the borders of the transcription bubble moved in approximate register with the transcript 3'-end. The backward movements of halted E. coli RNA polymerase are similar to a recently postulated model for discontinuous translocation during transcription, but differ from those reported for arrested RNA polymerase II transcription complexes.
The Escherichia coli GreA and GreB proteins induce cleavage of 3' fragments from nascent transcripts in halted transcription complexes. We have overproduced and purified the GreA protein and tested how it affects initiation, pausing, and termination by E. coli RNA polymerase. Recombinant GreA induced cleavage of two to three nucleotide fragments in two promoter-proximal complexes, whereas an apparently endogenous cleavage removed a single larger fragment. Both types of cleavage stopped once the transcript was shortened to approximately 10 nucleotides. However, during initiation, GreA induced cleavage of transcripts as short as four nucleotides, inhibiting their release as abortive products and stimulating both productive initiation and "primer-shifting" at a weak promoter. GreA induced repetitive cleavage over a long distance in complexes containing a long G-less nascent transcript. However, reverse translocation was inhibited in transcription complexes that contained a G-rich, C-less nascent transcript. Substituting IMP for GMP in the transcript relieved inhibition. Finally, GreA had little effect on transcription through the his and trp leader pause sites or on termination at nine different p-independent terminators. We propose that transcript cleavage and reverse translocation are controlled in part by backsliding of the nascent transcript through an RNA-binding site.
Streptolydigin (stl), a bacteriostatic inhibitor of transcription elongation, interacts with the beta subunit of Escherichia coli RNA polymerase. We have defined the target for stl resistance using chemical mutagenesis and mutagenic polymerase chain reaction. Mutations resulting in stl resistance are confined to a small cluster of contiguous amino acids, amino acids 543 to 546. These stlr mutants differ from one another in their levels of resistance to stl in vivo and in vitro. We have analyzed two of the mutants, A543V and F545S, for their effects on elongation and termination in vivo and in vitro. Neither affected termination at rho-dependent or rho-independent terminators. These mutants were indistinguishable from wild type in a T7 in vitro elongation assay. F545S, however, did exhibit slower elongation kinetics in a lambda tR1 pausing assay. We conclude that mutations in the stlr region can influence transcription elongation, but that these amino acids are not directly involved in catalysis.
A key feature of transcriptional attenuation in some amino acid biosynthetic operons is a transcriptional pause that occurs immediately after synthesis of the first leader transcript secondary structure. Both RNA secondary structure and downstream DNA sequence are important for pausing at these sites; however, the precise RNA structures involved and the relative contribution of other RNA and DNA bases to pausing are unknown. We studied the effects of base substitutions upstream from the his leader pause site (immediately prior to addition of G103) to determine how nucleic acid sequences and RNA structure contribute to pausing. By testing compensatory base substitutions, we found that pausing depended in part on an RNA secondary structure containing a five base-pair stem and eight nucleotide loop, which we call the his pause RNA hairpin. The his pause hairpin forms 11 nucleotides upstream from the paused transcript 3' end and thus corresponds to only the upper portion of the larger his A:B leader transcript secondary structure. Some base substitutions in the ten nucleotides between the pause hairpin and the 3' end of the transcript increased pausing, whereas others decreased pausing. However, compensatory substitutions that restored pairing of these bases in the lower portion of the A:B secondary structure did not alter these effects. Changing the 3'-terminal nucleotide of the transcript (U102) altered both the position and strength of pausing. Thus, in addition to the downstream DNA sequence, three distinct segments of nucleic acid upstream from the nucleotide-addition site in the transcription complex contribute to pausing in different ways: the pause RNA hairpin, the 3'-proximal region of transcript or DNA template, and the 3'-terminal nucleotide. We suggest that electrostatic interaction between the pause hairpin and RNA polymerase, rather than disruption of an RNA:DNA heteroduplex, delays elongation at the his leader pause site.
RNA polymerases pause conspicuously at certain positions on a DNA template. At the well-studied pause sites in the attenuation control regions that precede the trp and his operons, both formation of secondary structure in the nascent transcript and the DNA sequence immediately downstream contribute to pausing. The mechanisms of these effects are unknown. We report here studies on the structure of the RNA and DNA strands in purified trp and his paused transcription complexes in comparison to ten elongation complexes halted by nucleoside triphosphate deprivation. A 14 to 22 nucleotide region of the DNA strands was accessible to modification by KMnO4 or diethylpyrocarbonate in both the paused and halted transcription complexes. However, the region in front of the nucleotide-addition site was reactive only in some halted complexes. In both types of complexes, approximately eight nucleotides on the template strand immediately preceding the 3' end were protected from modification. We also examined the sensitivity of the nascent transcript to RNase A and found that the 3'-proximal eight nucleotide region could be cleaved without complete loss of the potential for elongation. However, a model RNA:DNA hybrid designed to mimic a hybrid in the transcription complex could also be cleaved under similar conditions. Together, the results suggest that the 3'-proximal eight nucleotides of transcript may pair with the DNA template and that this structure is not disrupted by hairpin formation at a pause site. Rather, pausing may result from distinct interactions between RNA polymerase and both the pause RNA hairpin and the downstream DNA sequence.
Transcriptional regulation of the human c-myc gene, an important aspect of cellular differentiation, occurs in part at the level of transcript elongation. In vivo, transcriptional arrest, due to either pausing or termination, occurs near the junction between the first exon and first intron and varies with the growth state of the cell. We have tested the transcription of c-myc templates in HeLa nuclear extracts. We did not observe significant arrest under standard conditions, but we found that a considerable fraction of transcription complexes stopped at the c-myc TII site (just past the first exon-intron junction) when the KCl concentration was raised to 400 mM during elongation. Transcriptional arrest at TII also was observed at KCl concentrations as low as 130 mM and when potassium acetate or potassium glutamate was substituted for KCl. Under these conditions, arrest occurred at the TII site when transcription was initiated at either the c-myc P2 promoter or the adenovirus 2 major late promoter. Further, the TII sequence itself, in forward but not reverse orientation, was sufficient to stop transcription in a HeLa nuclear extract. By separating the TII RNA from active transcription complexes by using gel filtration, we found that arrest at TII at 400 mM KCl resulted in transcript release and thus true transcriptional termination. The efficiency of termination at TII depended on the growth state of the cells from which the extracts were made, suggesting that some factor or factors control premature termination in c-myc.
The kinetics of transcription by Escherichia coli RNA polymerase relate directly to the regulation of transcription and to the properties of processive enzymes in general, but analysis of RNA polymerase movement along the DNA template has so far been limited to the study of populations of enzyme molecules. The ability to view nanometre-sized particles with the light microscope suggested a method of monitoring transcription by individual RNA polymerase molecules. We describe here the behaviour of 40-nm-diameter particles of colloidal gold attached to the ends of DNA molecules being transcribed by RNA polymerase immobilized on a glass surface. The tethered gold particles are released from the surface at times after addition of nucleoside triphosphates that are consistent with the kinetics of transcription by RNA polymerase in solution. Analysis of the brownian motion of the gold particles enabled us to measure the movement along the template DNA of individual polymerase molecules.
To test features of the current model of transcription attenuation in amino acid biosynthetic operons, alterations were introduced into the trp operon leader region and expression of the mutated operons was examined in miaA and miaA+ Escherichia coli strains that lacked the trp repressor. The miaA mutation prevents modification of the adenosine residue immediately 3' of the anticodon of tRNAs that interact with codons beginning with uridine. The undermodified tRNA(Trp) in miaA strains is thought to increase readthrough at the trp attenuator by slowing ribosome movement over two tandem Trp codons in the 14-codon leader peptide coding region. The rate of translation of these two "control codons" is thought to be the key step in determining the extent of transcription attenuation in the trp leader region. Sequential deletion of trpL DNA specifying the leader peptide initiation region, RNA segment 1, RNA segment 2 and RNA segment 3 alternately decreased and increased trp operon expression, a result consistent with previous findings in another bacterium and the generally accepted model for transcription attenuation. Replacement of the tandem Trp control codons by AGG-UGC (Arg-Cys) codons eliminated the miaA-dependent increase in transcription readthrough. Replacement of the Trp control codons by AGG-UGA (Arg-stop) codons caused complete readthrough at the trp attenuator as well as abolishing the miaA effect. Presumably, the ribosome terminating translation at the new UGA codon mimics the effect of a stalled ribosome at the Trp control codons. This finding suggests that ribosome dissociation at some stop codons is slow relative to the time required for transcription of the trp leader region. Thus, most ribosomes translating the trp leader peptide coding region may remain attached to the natural UGA stop codon until after the attenuation decision is made. The interpretation supports models for trp operon attenuation in which the elevated basal level readthrough is determined by occasional ribosome release prior to synthesis of the 3:4 terminator hairpin.
Escherichia coli RNA polymerase pauses immediately after transcription of certain sequences that can form stable secondary structures in the nascent RNA transcript; pausing appears to be essential for several types of bacterial transcription attenuation mechanisms. Because base changes that weaken the RNA secondary structures reduce the half-life of pausing by RNA polymerase, nascent transcript RNA hairpins are thought to cause pausing at these sites. We show here that, for the well characterized trpL pause site, the determinants of transcription pausing are not limited to the RNA hairpin, but include the not-yet-transcribed sequence of DNA immediately downstream from the pause site. We show that this effect extends to bases up to fourteen nucleotides downstream from the pause site, that placement of a oligo(dT) tract in the nontranscribed strand in this region does not convert the pause site to a termination site, and that shifting the position of pausing by one nucleotide downstream almost eliminates pausing. From an analysis of many variants of this downstream sequence, we argue that the effect of downstream sequence is not related simply to its GC content. We suggest that these effects are mediated by altered interactions between RNA polymerase and the DNA template downstream from the enzyme's active site.
Control of transcription at pause and termination sites is common in bacteria. Many transcriptional pause and termination events are thought to occur in response to formation of an RNA hairpin in the nascent transcript. Some mutations in the beta-subunit of Escherichia coli RNA polymerase that confer resistance to the transcription inhibitor rifampicin also alter the response to transcriptional pause and termination signals. Here, we report isolation of termination-altering mutations that do not confer rifampicin resistance and show that such mutations occur predominantly in limited regions of the beta-subunit polypeptide. One region is between amino acid residues 500 and 575, which encompasses the locations of almost all known rifampicin-resistance mutations. Many termination-altering mutations also occur in two other regions: between amino acid residues 740 and 840 and near the carboxyl terminus of the beta-subunit (amino acid residues 1225-1342). Amino acid sequences in these three regions of the beta-subunit are conserved between prokaryotic and eukaryotic beta-subunit homologs. Several mutations that alter transcription termination in vitro affect amino acid residues that are identical in prokaryotic and eukaryotic RNA polymerase beta-subunit homologs, suggesting that they alter an important function common to multisubunit RNA polymerases. We propose that these three regions of the beta-subunit may contact the nascent RNA transcript, the RNA-DNA heteroduplex, or the DNA template in the transcription complex and that mutations in these regions alter transcription pausing and termination by affecting these contacts.
The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined. Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport. Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes. These two proteins confer specificity on the LIV-I and LS transport systems. livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF. The protein products of the livHMGF genes are shared by the two systems. An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane. Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins. A deletion strain that does not express any of the liv genes was constructed. This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained.
A plasmid was constructed that overproduces the Escherichia coli RNA polymerase beta subunit from a lac promoter-rpoB fusion. The overproduced, plasmid-encoded beta subunit assembled into functional RNA polymerase that supplied greater than 90% of the transcriptional capacity of the cells. Excess beta subunit segregated into insoluble inclusion bodies and was not deleterious to cell growth. By insertion of a XhoI linker sequence (CTCGAG) and accompanying deletion of variable amounts of rpoB sequences, 13 structural alterations were isolated in the first and last thirds of the plasmid-borne rpoB gene. Twelve of these alterations appeared to reduce or prevent assembly of plasmid-encoded beta subunit into RNA polymerase. One alteration had no discernible effect on assembly or function of the beta subunit; eight others appeared to inhibit assembly but still produced detectable transcriptional activity. Three of these nine alterations produced beta-subunit polypeptides that inhibited cell growth at 32 degrees C, even though they were present in less than 50% of the cell RNA polymerase. When assembled into RNA polymerase, these three altered beta subunits apparently affected essential RNA polymerase functions. Four of the recovered alterations appeared to inhibit completely or almost completely assembly of the beta subunit into RNA polymerase. The results are consistent with a hypothesis that sequences in the first third of the beta-subunit polypeptide are especially important for proper folding and assembly of the beta subunit.
The Salmonella typhimurium his leader region contains a well documented transcription attenuator. We report here the results of in vitro transcription studies that characterized a transcription pause site in the his leader region. The pause occurred after synthesis of the first his leader secondary structure (A:B) and immediately preceding addition of G103 to the nascent transcript. RNA polymerase pausing at this site would allow a ribosome synthesizing the his leader peptide to release the paused polymerase and synchronize transcription and translation of the his leader region. The half-life of transcription complexes paused in the his leader was enhanced by NusA, but not guanosine 5'-diphosphate 3'-diphosphate. Nuclease digestion and RNA modeling studies were consistent with a compact three-dimensional structure for the his pause RNA. The half-life of the his leader paused transcription complex was decreased greatly on altered templates in which the C71-G93 base pair was disrupted but was unchanged when the C65-G100 base pair was disrupted. This result is consistent with a model for the structure of paused transcription complexes in which a portion of the RNA:DNA elongation heteroduplex is retained.
Transcription pausing is a key step in many prokaryotic transcription attenuation mechanisms. Pausing is thought to occur when an RNA hairpin forms near the 3' end of a growing transcript. We report here the isolation of the trp leader paused transcription complex containing a defined 92-nucleotide nascent transcript. Digestion of isolated paused complexes with RNase T1 suggests that the trp leader RNA hairpin designated 1:2 forms in the paused transcription complex. The transcription factor NusA alters the RNase T1 digestion pattern of the 92-nucleotide pause transcript in the complex but not the cleavage patterns of purified pause RNA, suggesting that NusA specifically affects the 1:2 hairpin in the paused transcription complex. The isolated paused transcription complex retains the ability to resume transcription. Kinetic studies on the resumption of elongation suggest that NusA is a non-competitive inhibitor of paused complex release and that the Ks for GTP is around 300 microM. RNA polymerase in the paused transcription complex protects approximately 30 base-pairs on both DNA strands from exonuclease digestion.
To determine whether RNA polymerase pauses during transcription in vivo, we have examined transcripts of the trp operon leader regions of Serratia marcescens and Escherichia coli. Labeled RNAs synthesized in E. coli strains containing plasmids bearing wild-type or mutant trp leader regions of S. marcescens or E. coli were isolated by hybridization and analyzed by polyacrylamide gel electrophoresis. The labeled RNAs synthesized in vivo on the S. marcescens wild-type and deletion mutant plasmids were the same size as the in vitro pause and leader transcripts. Hybridization of the presumed in vivo pause RNAs, and control in vitro pause RNAs, to M13 phage DNA containing a trp leader region deletion followed by treatment with S1 nuclease produced identical protected RNA species, proving that the in vitro and in vivo RNAs were identical. The amount of labeled pause RNAs relative to leader RNAs decreased following a chase with unlabeled uridine. E. coli RNAs identical to the previously characterized in vitro pause and leader transcripts were demonstrated by electrophoretic band position and fingerprint analysis. The finding that transcription pausing occurs in vivo is consistent with the view that transcription pausing and ribosome release of paused transcription complexes are responsible for the coupling of translation with transcription that is crucial to attenuation.
