We study fundamental issues in the molecular biology of gene expression in bacteria using cutting edge biochemical and genetic techniques. At present, we are focused primarily on the mechanism of transcription initiation, the architecture of transcription complexes, the relationship between structure and function of RNA polymerase (RNAP), and the mechanisms by which the transcription apparatus responds to signals from inside and outside of the cell. We have studied the control of ribosome synthesis in Escherichia coli, because this is so central to cell function, because rRNA promoters account for the majority of transcription in the cell, and because the genes for rRNA and other parts of the translation machinery display a fascinating and complex network of regulatory responses. In recent years we have discovered that the factors responsible for controlling rRNA synthesis also regulate hundreds of other genes, and we have begun to study the regulation of many of those genes as well. Current projects in the lab include studies on (i) the conformational changes in the promoter-RNAP complex that account for the tremendous variation in activity that occurs in response to differences in natural promoter sequences, (ii) the transcription factors ppGpp and DksA and their interactions with RNAP responsible for transcription regulation, and (iii) a transcription factor that facilitates RNA polymerase assembly. In addition, using tools of cell biology including fluorescence microscopy, we have discovered that bacteria contain nucleolus-like structures; i.e. rRNA operons come together in space even though the rRNA genes are spread out over 180 degrees of the circular E. coli chromosome. We are now exploring the factors that are responsible for these long-range interactions and their contributions to chromosome folding.
Microbiology 612: Prokaryotic Molecular Biology
Graduate Program in Cellular and Molecular Biology
Genetics Ph.D. Training Program
Microbiology Doctoral Training Program
Molecular Biosciences Training Grant Program
Biotechnology Training Program
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The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chromosome organization in Escherichia coli, we examined the relative positions of the ribosomal RNA (rRNA) operons in space. The seven rRNA operons are nearly identical and separated from each other by as much as 180° on the circular genetic map, a distance of ≥2 million base pairs. By inserting binding sites for fluorescent proteins adjacent to the rRNA operons and then examining their positions pairwise in live cells by epifluorescence microscopy, we found that all but rrnC are in close proximity. Colocalization of the rRNA operons required the rrn P1 promoter region but not the rrn P2 promoter or the rRNA structural genes and occurred with and without active transcription. Non-rRNA operon pairs did not colocalize, and the magnitude of their physical separation generally correlated with that of their genetic separation. Our results show that E. coli bacterial chromosome folding in three dimensions is not dictated entirely by genetic position but rather includes functionally related, genetically distant loci that come into close proximity, with rRNA operons forming a structure reminiscent of the eukaryotic nucleolus.
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Throughout the bacterial domain, the alarmone ppGpp dramatically reprograms transcription following nutrient limitation. This "stringent response" is critical for survival and antibiotic tolerance and is a model for transcriptional regulation by small ligands. We report that ppGpp binds to two distinct sites 60 Å apart on E. coli RNA polymerase (RNAP), one characterized previously (site 1) and a second identified here at an interface of RNAP and the transcription factor DksA (site 2). The location and unusual tripartite nature of site 2 account for the DksA-ppGpp synergism and suggest mechanisms for ppGpp enhancement of DksA's effects on RNAP. Site 2 binding results in the majority of ppGpp's effects on transcription initiation in vitro and in vivo, and strains lacking site 2 are severely impaired for growth following nutritional shifts. Filling of the two sites at different ppGpp concentrations would expand the dynamic range of cellular responses to changes in ppGpp levels.
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Multiple essential small GTPases are involved in the assembly of the ribosome or in the control of its activity. Among them, ObgE (CgtA) has been shown recently to act as a ribosome antiassociation factor that binds to ppGpp, a regulator whose best-known target is RNA polymerase. The present study was aimed at elucidating the expression of obgE in Escherichia coli We show that obgE is cotranscribed with ribosomal protein genes rplU and rpmA and with a gene of unknown function, yhbE We show here that about 75% of the transcripts terminate before obgE, because there is a transcriptional terminator between rpmA and yhbE As expected for ribosomal protein operons, expression was highest during exponential growth, decreased during entry into stationary phase, and became almost undetectable thereafter. Expression of the operon was derepressed in mutants lacking ppGpp or DksA. However, regulation by these factors appears to occur post-transcription initiation, since no effects of ppGpp and DksA on rplU promoter activity were observed in vitro The conserved and essential ObgE GTPase binds to the ribosome and affects its assembly. ObgE has also been reported to impact chromosome segregation, cell division, resistance to DNA damage, and, perhaps most interestingly, persister formation and antibiotic tolerance. However, it is unclear whether these effects are related to its role in ribosome formation. Despite its importance, no studies on ObgE expression have been reported. We demonstrate here that obgE is expressed from an operon encoding two ribosomal proteins, that the operon's expression varies with the growth phase, and that it is dependent on the transcription regulators ppGpp and DksA. Our results thus demonstrate that obgE expression is coupled to ribosomal gene expression.
Most Escherichia coli promoters initiate transcription with a purine 7 or 8 nt downstream from the -10 hexamer, but some promoters, including the ribosomal RNA promoter rrnB P1, start 9 nt from the -10 element. We identified promoter and RNA polymerase determinants of this noncanonical rrnB P1 start site using biochemical and genetic approaches including mutational analysis of the promoter, Fe(2+) cleavage assays to monitor template strand positions near the active-site, and Bpa cross-linking to map the path of open complex DNA at amino acid and nucleotide resolution. We find that mutations in several promoter regions affect transcription start site (TSS) selection. In particular, we show that the absence of strong interactions between the discriminator region and σ region 1.2 and between the extended -10 element and σ region 3.0, identified previously as a determinant of proper regulation of rRNA promoters, is also required for the unusual TSS. We find that the DNA in the single-stranded transcription bubble of the rrnB P1 promoter complex expands and is "scrunched" into the active site channel of RNA polymerase, similar to the situation in initial transcribing complexes. However, in the rrnB P1 open complex, scrunching occurs before RNA synthesis begins. We find that the scrunched open complex exhibits reduced abortive product synthesis, suggesting that scrunching and unusual TSS selection contribute to the extraordinary transcriptional activity of rRNA promoters by increasing promoter escape, helping to offset the reduction in promoter activity that would result from the weak interactions with σ.
In bacterial transcription initiation, RNA polymerase (RNAP) selects a transcription start site (TSS) at variable distances downstream of core promoter elements. Using next-generation sequencing and unnatural amino acid-mediated protein-DNA cross-linking, we have determined, for a library of 4(10) promoter sequences, the TSS, the RNAP leading-edge position, and the RNAP trailing-edge position. We find that a promoter element upstream of the TSS, the "discriminator," participates in TSS selection, and that, as the TSS changes, the RNAP leading-edge position changes, but the RNAP trailing-edge position does not change. Changes in the RNAP leading-edge position, but not the RNAP trailing-edge position, are a defining hallmark of the "DNA scrunching" that occurs concurrent with RNA synthesis in initial transcription. We propose that TSS selection involves DNA scrunching prior to RNA synthesis.
The central metabolite acetyl phosphate (acP) has long been proposed to influence transcription regulation by directly transferring its phosphoryl group to a number of response regulators in many bacterial species. Here, we provide in vitro evidence for this proposition and demonstrate, using an in vitro transcription system, that acP-dependent phosphorylation of aspartate 51 of CpxR induces transcription of one of its regulon members in E. coli, cpxP. We also used this in vitro transcription system to extend our previously reported in vivo data that hypothesized that acetylation of RNA polymerase (RNAP) influences acP-dependent cpxP transcription, using glutamine as a genetic mimic for acetylated arginine 291 of the carboxy-terminal domain of RNAP α subunit. The data we present here lend strong support to the hypothesis that acP has a direct effect on transcription regulation in E. coli via phosphorylation of CpxR, and that RNAP acetylation can modulate this response.
RNA polymerase binds tightly to DNA to recognize promoters with high specificity but then releases these contacts during the initial stage of transcription. We report a site-specific crosslinking approach to map the DNA path in bacterial transcription intermediates at amino acid and nucleotide resolution. After validating the approach by showing that the DNA path in open complexes (RPO) is the same as in high-resolution X-ray structures, we define the path following substrate addition in "scrunched" complexes (RPITC). The DNA bulges that form within the transcription bubble in RPITC are positioned differently on the two strands. Our data suggest that the non-template strand bulge is extruded into solvent in complexes containing a 5-mer RNA, whereas the template strand bulge remains within the template strand tunnel, exerting stress on interactions between the β flap, β' clamp, and σ3.2. We propose that this stress contributes to σ3.2 displacement from the RNA exit channel, facilitating promoter escape.
Transcription by RNA polymerase (RNAP) in bacteria requires specific promoter recognition by σ factors. The major variant σ factor (σ(54)) initially forms a transcriptionally silent complex requiring specialized adenosine triphosphate-dependent activators for initiation. Our crystal structure of the 450-kilodalton RNAP-σ(54) holoenzyme at 3.8 angstroms reveals molecular details of σ(54) and its interactions with RNAP. The structure explains how σ(54) targets different regions in RNAP to exert its inhibitory function. Although σ(54) and the major σ factor, σ(70), have similar functional domains and contact similar regions of RNAP, unanticipated differences are observed in their domain arrangement and interactions with RNAP, explaining their distinct properties. Furthermore, we observe evolutionarily conserved regulatory hotspots in RNAPs that can be targeted by a diverse range of mechanisms to fine tune transcription.
A cross-campus, cross-career stage and cross-disciplinary series of discussions at a large public university has produced a series of recommendations for addressing the problems confronting the biomedical research community in the US.
Horizontal gene transfer by conjugation plays a major role in bacterial evolution, allowing the acquisition of new traits, such as virulence and resistance to antibacterial agents. With the increased antibiotic resistance in bacterial pathogens, a better understanding of how bacteria modulate conjugation under changing environments and the genetic factors involved is needed. Despite the evolutionary advantages conjugation may confer, the process can be quite stressful for the donor cell. Here, we characterize the ability of TraR, encoded on the episomal F' plasmid, to upregulate the σ(E) extracytoplasmic stress pathway in Escherichia coli. TraR, a DksA homolog, modulates transcription initiation through the secondary channel of RNA polymerase. We show here that TraR activates transcription directly; however, unlike DksA, it does so without using ppGpp as a cofactor. TraR expression can stimulate the σ(E) extracytoplasmic stress response independently of the DegS/RseA signal transduction cascade. In the absence of TraR, bacteria carrying conjugative plasmids become more susceptible to external stress. We propose that TraR increases the concentrations of periplasmic chaperones and proteases by directly activating the transcription of σ(E)-dependent promoters; this increased protein folding capacity may prepare the bacterium to endure the periplasmic stress of sex pilus biosynthesis during mating.
Bacteria utilize multiple sigma factors that associate with core RNA polymerase (RNAP) to control transcription in response to changes in environmental conditions. In Escherichia coli and Salmonella enterica, Crl positively regulates the σ(S) regulon by binding to σ(S) to promote its association with core RNAP. We recently characterized the determinants in σ(S) responsible for specific binding to Crl. However, little is known about the determinants in Crl required for this interaction. Here, we present the X-ray crystal structure of a Crl homolog from Proteus mirabilis in conjunction with in vivo and in vitro approaches that probe the Crl-σ(S) interaction in E. coli. We show that the P. mirabilis, Vibrio harveyi, and E. coli Crl homologs function similarly in E. coli, indicating that Crl structure and function are likely conserved throughout gammaproteobacteria. We utilize phylogenetic conservation and bacterial two-hybrid analyses to predict residues in Crl important for the interaction with σ(S). The results of p-benzoylphenylalanine (BPA)-mediated UV cross-linking studies further support the model in which an evolutionarily conserved central cleft is the surface on Crl that binds to σ(S). Within this conserved binding surface, we identify a key residue in Crl that is critical for activation of Eσ(S)-dependent transcription in vivo and in vitro. Our study provides a physical basis for understanding the σ(S)-Crl interaction.
ABSTRACT DksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the "stringent response" to nutrient starvation in the gammaproteobacterium Escherichia coli and for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly related Rhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length to E. coli DksA but lacks the Zn finger motif of the E. coli DksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of an E. coli strain lacking the dksA gene and modulates transcription in vitro with E. coli RNA polymerase (RNAP) similarly to E. coli DksA. RSP2654 reduces RNAP-promoter complex stability in vitro with RNAPs from E. coli or R. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity to E. coli DksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsp has distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp. IMPORTANCE The role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis in Rhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.
Bacteria use multiple sigma factors to coordinate gene expression in response to environmental perturbations. In Escherichia coli and other γ-proteobacteria, the transcription factor Crl stimulates σ(S)-dependent transcription during times of cellular stress by promoting the association of σ(S) with core RNA polymerase. The molecular basis for specific recognition of σ(S) by Crl, rather than the homologous and more abundant primary sigma factor σ(70), is unknown. Here we use bacterial two-hybrid analysis in vivo and p-benzoyl-phenylalanine cross-linking in vitro to define the features in σ(S) responsible for specific recognition by Crl. We identify residues in σ(S) conserved domain 2 (σ(S)2) that are necessary and sufficient to allow recognition of σ(70) conserved domain 2 by Crl, one near the promoter-melting region and the other at the position where a large nonconserved region interrupts the sequence of σ(70). We then use luminescence resonance energy transfer to demonstrate directly that Crl promotes holoenzyme assembly using these specificity determinants on σ(S). Our results explain how Crl distinguishes between sigma factors that are largely homologous and activates discrete sets of promoters even though it does not bind to promoter DNA.
In Escherichia coli, FadR and FabR are transcriptional regulators that control the expression of fatty acid degradation and unsaturated fatty acid synthesis genes, depending on the availability of fatty acids. In this report, we focus on the dual transcriptional regulator FadR. In the absence of fatty acids, FadR represses the transcription of fad genes required for fatty acid degradation. However, FadR is also an activator, stimulating transcription of the products of the fabA and fabB genes responsible for unsaturated fatty acid synthesis. In this study, we show that FadR directly activates another fatty acid synthesis promoter, PfabH, which transcribes the fabHDG operon, indicating that FadR is a global regulator of both fatty acid degradation and fatty acid synthesis. We also demonstrate that ppGpp and its cofactor DksA, known primarily for their role in regulation of the synthesis of the translational machinery, directly inhibit transcription from the fabH promoter. ppGpp also inhibits the fadR promoter, thereby reducing transcription activation of fabH by FadR indirectly. Our study shows that both ppGpp and FadR have direct roles in the control of fatty acid promoters, linking expression in response to both translation activity and fatty acid availability.
Biofilm formation in Bacillus subtilis requires expression of the eps and tapA-sipW-tasA operons to synthesize the extracellular matrix components, extracellular polysaccharide and TasA amyloid proteins, respectively. Expression of both operons is inhibited by the DNA-binding protein master regulator of biofilm formation SinR and activated by the protein RemA. Here we show that RemA is a DNA-binding protein that binds to multiple sites upstream of the promoters of both operons and is both necessary and sufficient for transcriptional activation in vivo and in vitro. We further show that SinR negatively regulates eps operon expression by occluding RemA binding and thus for the P(eps) promoter SinR functions as an anti-activator. Finally, transcriptional profiling indicated that RemA was primarily a regulator of the extracellular matrix genes, but it also activated genes involved in osmoprotection, leading to the identification of another direct target, the opuA operon.
The global regulatory nucleotide ppGpp ("magic spot") regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp's target site on RNAP, and therefore its mechanism of action, has remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and β', and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.
Escherichia coli DksA is a transcription factor that binds to RNA polymerase (RNAP) without binding to DNA, destabilizing RNAP-promoter interactions, sensitizing RNAP to the global regulator ppGpp, and regulating transcription of several hundred target genes, including those encoding rRNA. Previously, we described promoter sequences and kinetic properties that account for DksA's promoter specificity, but how DksA exerts its effects on RNAP has remained unclear. To better understand DksA's mechanism of action, we incorporated benzoyl-phenylalanine at specific positions in DksA and mapped its cross-links to RNAP, constraining computational docking of the two proteins. The resulting evidence-based model of the DksA-RNAP complex as well as additional genetic and biochemical approaches confirmed that DksA binds to the RNAP secondary channel, defined the orientation of DksA in the channel, and predicted a network of DksA interactions with RNAP that includes the rim helices and the mobile trigger loop (TL) domain. Engineered cysteine substitutions in the TL and DksA coiled-coil tip generated a disulfide bond between them, and the interacting residues were absolutely required for DksA function. We suggest that DksA traps the TL in a conformation that destabilizes promoter complexes, an interaction explaining the requirement for the DksA tip and its effects on transcription.
Escherichia coli DksA works in conjunction with the small-molecule ppGpp to regulate transcription initiation negatively or positively, depending on the identity of the promoter. DksA is in a class of transcription factors that do not bind directly to DNA such as classical repressors or activators but rather bind in the RNA polymerase (RNAP) secondary channel such as the transcription elongation factors GreA and GreB in E. coli and TFIIS in eukaryotes. We found that substitution for either of two residues in its coiled-coil tip, D74 or A76, eliminates DksA function without affecting its apparent affinity for RNAP. The properties of DksA-Gre factor chimeras indicated that the coiled-coil tip is responsible for the DksA-specific effects on open complex formation. A conservative substitution at position 74, D74E, resulted in a loss of DksA function in both negative and positive control, and an E44D substitution at the analogous position in GreA resulted in a gain of function in both negative and positive control. That a single methylene group has such an extraordinary effect on these transcription factors highlights the critical nature of the identity of coiled-coil tip interactions with RNAP for open complex formation.
DksA is an RNA polymerase (RNAP) binding transcription factor that controls expression of a large number of genes in concert with the small-molecule "alarmone" ppGpp. DksA also aids in the resolution of conflicts between RNAP and DNA polymerase (DNAP) during genome replication. DksA was originally identified as a multicopy suppressor of the temperature sensitivity caused by deletion of the genes coding for the DnaKJ chaperone system. Here, we address a longstanding question regarding the role of DksA in ΔdnaKJ suppression. We demonstrate that DksA expression from a multicopy plasmid is necessary and sufficient for suppression, that overexpression occurs despite the fact that the major dksA promoter is feedback regulated in wild-type cells, and that weak, non-feedback-regulated transcription originating upstream of the major promoter for the dksA gene accounts for overexpression. We tentatively rule out three potential explanations for suppression related to known functions of DnaKJ. Because a determinant in DksA needed for the regulation of transcription initiation, but not for resolution of RNAP-DNAP conflicts, is needed to bypass the need for DnaKJ, we suggest that suppression results from an unidentified product whose promoter is directly or indirectly regulated by DksA.
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.
Escherichia coli DksA is an RNA polymerase (RNAP)-binding protein required for the regulation of a number of promoters, including those for the biosynthesis of rRNA, many ribosomal proteins, components of the flagellum, and several amino acids. Previous work demonstrated that DksA protein levels do not vary greatly in different growth conditions. We show here that DksA is a stable protein whose levels are kept constant by a negative feedback loop in which transcription from the dksA promoter is inhibited by DksA protein in conjunction with its cofactor ppGpp. We map the primary dksA promoter by primer extension, show that its activity increases in a strain lacking DksA, that the DksA protein accumulates when expressed from an exogenous promoter, that inhibition of transcription by DksA is direct since it occurs with purified components in vitro, and that inhibition correlates with effects of DksA on the lifetime of the dksA promoter complex. This work provides a mechanistic basis for the maintenance of constant cellular levels of DksA in E. coli and supports the model that transcription regulation by ppGpp/DksA derives from fluctuations in the concentrations of the small molecule cofactor rather than of DksA itself.
We show here that the promoters for many of the Escherichia coli ribosomal protein operons are regulated directly by two transcription factors, the small RNA polymerase-binding protein DksA and the nutritional stress-induced nucleotide ppGpp. ppGpp and DksA work together to inhibit transcription initiation from ribosomal protein promoters in vitro and in vivo. The degree of promoter regulation by ppGpp/DksA varies among the r-protein promoters, but some are inhibited almost as much as rRNA promoters. Thus, many r-protein operons are regulated at the level of transcription in addition to their control by the classic translational feedback systems discovered ~30 y ago. We conclude that direct control of r-protein promoters and rRNA promoters by the same signal, ppGpp/DksA, makes a major contribution to the balanced and coordinated synthesis rates of all of the ribosomal components.
We report an improved procedure for purification of the omega subunit of Escherichia coli RNA polymerase. In contrast to the original procedure, the revised procedure: (i) allows purification of omega entirely from the soluble fraction, obviating the need for denaturation/renaturation, (ii) results in >99% pure omega in only two chromatographic steps, and (iii) improves the yield of purified omega by at least 5-fold. Reconstitution of E. coli RNAP from omega purified by this procedure, as well as purified sigma and core RNAP lacking omega, produces active holoenzyme in vitro, and co-overexpression of omega from a plasmid containing rpoZ and an additional plasmid encoding the other RNAP core subunits results in production of active core enzyme in vivo.
The components of the Escherichia coli flagella apparatus are synthesized in a three-level transcriptional cascade activated by the master regulator FlhDC. The cascade co-ordinates the synthesis rates of a large number of gene products with each other and with nutritional conditions. Recent genome-wide studies have reported that flagellar transcription is altered in cells lacking the transcription regulators DksA or ppGpp, but some or all reported effects could be indirect, and some are contradictory. We report here that the activities of promoters at all three levels of the cascade are much higher in strains lacking dksA, resulting in overproduction of flagellin and hyperflagellated cells. In vitro, DksA/ppGpp inhibits the flhDC promoter and the sigma(70)-dependent fliA promoter transcribing the gene for sigma(28). However, DksA and ppGpp do not affect the sigma(28)-dependent fliA promoter or the sigma(28)-dependent fliC promoter in vitro, suggesting that the dramatic effects on expression of those genes in vivo are mediated indirectly through direct effects of DksA/ppGpp on FlhDC and sigma(28) expression. We conclude that DksA/ppGpp inhibits expression of the flagellar cascade during stationary phase and following starvation, thereby co-ordinating flagella and ribosome assembly and preventing expenditure of scarce energy resources on synthesis of two of the cell's largest macromolecular complexes.
The transcription factor DksA binds in the secondary channel of RNA polymerase (RNAP) and alters transcriptional output without interacting with DNA. Here we present a quantitative assay for measuring DksA binding affinity and illustrate its utility by determining the relative affinities of DksA for three different forms of RNAP. Whereas the apparent affinities of DksA for RNAP core and holoenzyme are the same, the apparent affinity of DksA for RNAP decreases almost 10-fold in an open complex. These results suggest that the conformation of RNAP present in an open complex is not optimal for DksA binding and that DNA directly or indirectly alters the interface between the two proteins.
At specific times during bacterial growth, the transcription factor DksA and the unusual nucleotide regulator ppGpp work synergistically to inhibit some Escherichia coli promoters (e.g. rRNA promoters) and to stimulate others (e.g. promoters for amino-acid synthesis and transport). However, the mechanism of DksA action remains uncertain, in part because DksA does not function like conventional transcription factors. To gain insights into DksA function, we identified mutations in dksA that bypassed the requirement for ppGpp by selecting for growth of cells lacking ppGpp on minimal medium without amino acids. We show here that two substitutions in DksA, L15F and N88I, result in higher DksA activity both in vivo and in vitro, primarily by increasing the apparent affinity of DksA for RNA polymerase (RNAP). The mutant DksA proteins suggest potential roles for ppGpp in DksA function, identify potential surfaces on DksA crucial for RNAP binding, and provide tools for future studies to elucidate the mechanism of DksA action.
The Escherichia coli DksA protein inserts into the RNA polymerase (RNAP) secondary channel, modifying the transcription initiation complex so that promoters with specific kinetic characteristics are regulated by changes in the concentrations of ppGpp and NTPs. We used footprinting assays to determine the specific kinetic intermediate, RP(I), on which DksA acts. Genetic approaches identified substitutions in the RNAP switch regions, bridge helix, and trigger loop that mimicked, reduced, or enhanced DksA function on rRNA promoters. Our results indicate that DksA binding in the secondary channel of RP(I) disrupts interactions with promoter DNA at least 25 A away, between positions -6 and +6 (the transcription start site is +1). We propose a working model in which the trigger loop and bridge helix transmit effects of DksA to the switch region(s), allosterically affecting switch residues that control clamp opening/closing and/or that interact directly with promoter DNA. DksA thus inhibits the transition to RP(I). Our results illustrate in mechanistic terms how transcription factors can regulate initiation promoter-specifically without interacting directly with DNA.
Bacterial promoter identification and characterization is not as straightforward as one might presume. Promoters vary widely in their similarity to the consensus recognition element sequences, in their activities, and in their utilization of transcription factors, and multiple approaches often must be used to provide a framework for understanding promoter regulation. Characterization of RNA polymerase-promoter complex formation in the absence of additional regulatory factors (basal promoter function) can provide a basis for understanding the steps in transcription initiation that are ultimately targeted by nutritional or environmental factors. Promoters can be localized using genetic approaches in vivo, but the detailed properties of the RNAP-promoter complex are studied most productively in vitro. We first describe approaches for identification of bacterial promoters and transcription start sites in vivo, including promoter-reporter fusions and primer-extension. We then describe a number of methods for characterization of RNAP-promoter complexes in vitro, including in vitro transcription, gel mobility shift assays, footprinting, and filter binding. Utilization of these methods can result in determination of not only basal promoter strength but also the rates of transcription initiation complex formation and decay.
Sequence-based searches identified a new family of genes in proteobacteria, named rnk, which shares high sequence similarity with the C-terminal domains of the Gre factors (GreA and GreB) and the Thermus/Deinococcus anti-Gre factor Gfh1. We solved the X-ray crystal structure of Escherichia coli regulator of nucleoside kinase (Rnk) at 1.9 A resolution using the anomalous signal from the native protein. The Rnk structure strikingly resembles those of E. coli GreA and GreB and Thermus Gfh1, all of which are RNA polymerase (RNAP) secondary channel effectors and have a C-terminal domain belonging to the FKBP fold. Rnk, however, has a much shorter N-terminal coiled coil. Rnk does not stimulate transcript cleavage in vitro, nor does it reduce the lifetime of the complex formed by RNAP on promoters. We show that Rnk competes with the Gre factors and DksA (another RNAP secondary channel effector) for binding to RNAP in vitro, and although we found that the concentration of Rnk in vivo was much lower than that of DksA, it was similar to that of GreB, consistent with a potential regulatory role for Rnk as an anti-Gre factor.
Early work identified two promoter regions, the -10 and -35 elements, that interact sequence specifically with bacterial RNA polymerase (RNAP). However, we now know that several additional promoter elements contact RNAP and influence transcription initiation. Furthermore, our picture of promoter control has evolved beyond one in which regulation results solely from activators and repressors that bind to DNA sequences near the RNAP binding site: many important transcription factors bind directly to RNAP without binding to DNA. These factors can target promoters by affecting specific kinetic steps on the pathway to open complex formation, thereby regulating RNA output from specific promoters.
We recently proposed that a nontemplate strand base in the discriminator region of bacterial promoters, the region between the -10 element and the transcription start site, makes sequence-specific contacts to region 1.2 of the sigma subunit of Escherichia coli RNA polymerase (RNAP). Because rRNA promoters contain sequences within the discriminator region that are suboptimal for interaction with sigma1.2, these promoters have the kinetic properties required for regulation by the RNAP-binding factors DksA and ppGpp. Here, we use zero-length cross-linking and mutational, kinetic, and footprinting studies to map RNAP interactions with the nontemplate strand bases at the junction of the -10 element and the discriminator region in an unregulated rRNA promoter variant and in the lambdaP(R) promoter. Our studies indicate that nontemplate strand bases adjacent to the -10 element bind within a 9-aa interval in sigma1.2 (residues 99-107). We also demonstrate that the downstream-most base on the nontemplate strand of the -10 hexamer cross-links to sigma region 2, and not to sigma1.2. Our results refine models of RNAP-DNA interactions in the promoter complex that are crucial for regulation of transcription initiation.
Identification of the RNA polymerase (RNAP) binding site for ppGpp, a central regulator of bacterial transcription, is crucial for understanding its mechanism of action. A recent high-resolution X-ray structure defined a ppGpp binding site on Thermus thermophilus RNAP. We report here effects of ppGpp on 10 mutant Escherichia coli RNAPs with substitutions for the analogous residues within 3-4 A of the ppGpp binding site in the T. thermophilus cocrystal. None of the substitutions in E. coli RNAP significantly weakened its responses to ppGpp. This result differs from the originally reported finding of a substitution in E. coli RNAP eliminating ppGpp function. The E. coli RNAPs used in that study likely lacked stoichiometric amounts of omega, an RNAP subunit required for responses of RNAP to ppGpp, in part explaining the discrepancy. Furthermore, we found that ppGpp did not inhibit transcription initiation by T. thermophilus RNAP in vitro or shorten the lifetimes of promoter complexes containing T. thermophilus RNAP, in contrast to the conclusion in the original report. Our results suggest that the ppGpp binding pocket identified in the cocrystal is not the one responsible for regulation of E. coli ribosomal RNA transcription initiation and highlight the importance of inclusion of omega in bacterial RNAP preparations.
One of the major signalling pathways responsible for intercompartmental communication between the cell envelope and cytoplasm in Escherichia coli is mediated by the alternative sigma factor, sigmaE. sigmaE has been studied primarily for its role in response to the misfolding of outer membrane porins. This response is essentially reactionary; cells are stressed, porin folding is disrupted, and the response is activated. sigmaE can also be activated following starvation for a variety of nutrients by the alarmone ppGpp. This response is proactive, as sigmaE is activated in the absence of any obvious damage to the cell envelope sensed by the stress signalling pathway. Here we examine the mechanism of regulation of sigmaE by ppGpp. ppGpp has been proposed to activate at least two alternative sigma factors, sigmaN and sigmaS, indirectly by altering the competition for core RNA polymerase between the alternative sigma factors and the housekeeping sigma factor, sigma70. In vivo experiments with sigmaE are consistent with this model. However, ppGpp and its cofactor DksA can also activate transcription by EsigmaEin vitro, suggesting that the effects of ppGpp on sigmaE activity are both direct and indirect.
A major challenge in systems biology is to integrate our mechanistic understanding of gene regulation to predict quantitatively how cells will respond to environmental changes. Living cells respond rapidly to the availability of nutrients in part by altering production of ribosomal RNA (rRNA), a limiting component in the biosynthesis of ribosomes. Studies of rRNA transcription by the RNA polymerase of Escherichia coli have identified regulatory roles for guanosine tetraphosphate (ppGpp), the initiating nucleotide, and the protein DksA. To what extent findings from in vitro studies can be used to quantitatively predict in vivo responses to changing nutrient environments is unknown. We developed a mechanistic mathematical model for rRNA transcriptional responses to such changes. Our model accounts for binding of RNAP to its rRNA promoter to form a closed complex, isomerization from a closed complex to an open complex, reversible incorporation of the initiating NTP (iNTP), transcript elongation, and clearance of the promoter. Further, the model incorporates interactions between ppGpp and DksA with transcription intermediates, and it includes an empirical correction to account for salt effects. The model biophysical parameters were determined using 33 single- and multi-round transcription experiments spanning 487 in vitro measurements. By incorporating in vivo measurements of ppGpp and ATP, the model correctly predicted rRNA production rates for cellular responses to nutrient upshifts, downshifts, and outgrowth into fresh medium. Inclusion of DksA was essential in all three cases. Our work provides a foundation for using data-driven computational models to predict the kinetics of in vivo transcriptional responses.
The bacterial signaling molecules ppGpp and pppGpp regulate transcription initiation in response to starvation by altering RNA polymerase activity. In this issue, Wang et al. (2007) show that (p)ppGpp also inhibits DNA replication elongation by interfering with DNA primase activity. Halting replication may help cells to maintain genomic integrity during periods of transient nutrient limitation.
Escherichia coli DksA, GreA, and GreB have similar structures and bind to the same location on RNA polymerase (RNAP), the secondary channel. We show that GreB can fulfil some roles of DksA in vitro, including shifting the promoter-open complex equilibrium in the dissociation direction, thus allowing rRNA promoters to respond to changes in the concentration of ppGpp and NTPs. However, unlike deletion of the dksA gene, deletion of greB had no effect on rRNA promoters in vivo. We show that the apparent affinities of DksA and GreB for RNAP are similar, but the cellular concentration of GreB is much lower than that of DksA. When over-expressed and in the absence of competing GreA, GreB almost completely complemented the loss of dksA in control of rRNA expression, indicating its inability to regulate rRNA transcription in vivo results primarily from its low concentration. In contrast to GreB, the apparent affinity of GreA for RNAP was weaker than that of DksA, GreA affected rRNA promoters only modestly in vitro and, even when over-expressed, GreA did not affect rRNA transcription in vivo. Thus, binding in the secondary channel is necessary but insufficient to explain the effect of DksA on rRNA transcription. Neither Gre factor was capable of fulfilling two other functions of DksA in transcription initiation: co-activation of amino acid biosynthetic gene promoters with ppGpp and compensation for the loss of the omega subunit of RNAP in the response of rRNA promoters to ppGpp. Our results provide important clues to the mechanisms of both negative and positive control of transcription initiation by DksA.
CTXPhi is a Vibrio cholerae-specific temperate filamentous phage that encodes cholera toxin. CTXPhi lysogens can be induced with DNA damage-inducing agents such as UV light, leading to the release of CTXPhi virions and the rapid dissemination of cholera toxin genes to new V. cholerae hosts. This environmental regulation is directly mediated by LexA, the host-encoded global SOS transcription factor. LexA and a phage-encoded repressor, RstR, both repress transcription from P(rstA), the primary CTXPhi promoter. Because the LexA binding site is located upstream of the core P(rstA) promoter and overlaps with A-tract sequences, we speculated that LexA represses P(rstA) by occluding a promoter UP element, a binding site for the C-terminal domain of the alpha subunit of RNA polymerase (RNAP) (alphaCTD). Using in vitro transcription assays, we have shown that the LexA binding site stimulates maximal rstA transcription in the absence of any added factors. The alphaCTD of RNAP is required for this stimulation, demonstrating that the LexA site contains, or overlaps with, a promoter UP element. LexA represses rstA transcription by normal RNAP but fails to repress rstA transcription catalyzed by RNAP lacking the alphaCTD. DNase I footprint analysis mapped the alphaCTD binding site to the upstream promoter region that includes the LexA binding site. The addition of free alpha subunits blocked the binding of LexA to rstA promoter DNA, indicating that LexA and the alphaCTD directly compete for binding to their respective sites. To our knowledge, this is the first report of a repressor blocking transcription initiation by occluding a promoter UP element.
The Escherichia coli Crl protein has been described as a transcriptional coactivator for the stationary-phase sigma factor sigma(S). In a transcription system with highly purified components, we demonstrate that Crl affects transcription not only by the Esigma(S) RNA polymerase holoenzyme but also by Esigma(70) and Esigma(32). Crl increased transcription dramatically but only when the sigma concentration was low and when Crl was added to sigma prior to assembly with the core enzyme. Our results suggest that Crl facilitates holoenzyme formation, the first positive regulator identified with this mechanism of action.
DksA is a critical transcription factor in Escherichia coli that binds to RNA polymerase and potentiates control of rRNA promoters and certain amino acid promoters. Given the kinetic similarities between rRNA promoters and the fis promoter (Pfis), we investigated the possibility that DksA might also control transcription from Pfis. We show that the absence of dksA extends transcription from Pfis well into the late logarithmic and stationary growth phases, demonstrating the importance of DksA for growth phase-dependent regulation of fis. We also show that transcription from Pfis increases with steady-state growth rate and that dksA is absolutely required for this regulation. In addition, both DksA and ppGpp are required for inhibition of Pfis promoter activity following amino acid starvation, and these factors act directly and synergistically to negatively control Pfis transcription in vitro. DksA decreases the half-life of the intrinsically short-lived fis promoter-RNA polymerase complex and increases its sensitivity to the concentration of CTP, the predominant initiating nucleotide triphosphate for this promoter. This work extends our understanding of the multiple factors controlling fis expression and demonstrates the generality of the DksA requirement for regulation of kinetically similar promoters.
No abstract available.
Regulation of transcription initiation is generally attributable to activator/repressor proteins that bind to specific DNA sequences. However, regulators can also achieve specificity by binding directly to RNA polymerase (RNAP) and exploiting the kinetic variation intrinsic to different RNAP-promoter complexes. We report here a previously unknown interaction with Escherichia coli RNAP that defines an additional recognition element in bacterial promoters. The strength of this sequence-specific interaction varies at different promoters and affects the lifetime of the complex with RNAP. Selection of rRNA promoter mutants forming long-lived complexes, kinetic analyses of duplex and bubble templates, dimethylsulfate footprinting, and zero-Angstrom crosslinking demonstrated that sigma subunit region 1.2 directly contacts the nontemplate strand base two positions downstream of the -10 element (within the "discriminator" region). By making a nonoptimal sigma1.2-discriminator interaction, rRNA promoters create the short-lived complex required for specific responses to the RNAP binding factors ppGpp and DksA, ultimately accounting for regulation of ribosome synthesis.
Previous studies have come to conflicting conclusions about the requirement for the omega subunit of RNA polymerase in bacterial transcription regulation. We demonstrate here that purified RNAP lacking omega does not respond in vitro to the effector of the stringent response, ppGpp. DksA, a transcription factor that works in concert with ppGpp to regulate rRNA expression in vivo and in vitro, fully rescues the ppGpp-unresponsiveness of RNAP lacking omega, likely explaining why strains lacking omega display a stringent response in vivo. These results demonstrate that omega plays a role in RNAP function (in addition to its previously reported role in RNAP assembly) and highlight the importance of inclusion of omega in RNAP purification protocols. Furthermore, these results suggest that either one or both of two short segments in the beta' subunit that physically link omega to the ppGpp-binding region of the enzyme may play crucial roles in ppGpp and DksA function.
Amino acid starvation in Escherichia coli results in a spectrum of changes in gene expression, including inhibition of rRNA and tRNA promoters and activation of certain promoters for amino acid biosynthesis and transport. The unusual nucleotide ppGpp plays an important role in both negative and positive regulation. Previously, we and others suggested that positive effects of ppGpp might be indirect, resulting from the inhibition of rRNA transcription and, thus, liberation of RNA polymerase for binding to other promoters. Recently, we showed that DksA binds to RNA polymerase and greatly enhances direct effects of ppGpp on the negative control of rRNA promoters. This conclusion prompted us to reevaluate whether ppGpp might also have a direct role in positive control. We show here that ppGpp greatly increases the rate of transcription initiation from amino acid promoters in a purified system but only when DksA is present. Activation occurs by stimulation of the rate of an isomerization step on the pathway to open complex formation. Consistent with the model that ppGpp/DksA stimulates amino acid promoters both directly and indirectly in vivo, cells lacking dksA fail to activate transcription from the hisG promoter after amino acid starvation. Our results illustrate how transcription factors can positively regulate transcription initiation without binding DNA, demonstrate that dksA directly affects promoters in addition to those for rRNA, and suggest that some of the pleiotropic effects previously associated with dksA might be ascribable to direct effects of dksA on promoters involved in a wide variety of cellular functions.
The C-terminal domains of the two alpha-subunits (alphaCTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters. These interactions can increase transcription dramatically. Previously, effects of upstream DNA-alphaCTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements. However, contributions of nonspecific upstream DNA-alphaCTD interactions to promoter activity have not been evaluated extensively. Here, we examine effects of removal of alphaCTD, upstream promoter DNA, or both on the rate of open-complex formation with promoters that lack UP elements. Deletion of alphaCTD decreased the composite second-order association rate constant, k(a), of RNAP for the lacUV5 promoter by approximately 10-fold. Much of this effect was attributable to a decrease in the isomerization rate constant, k(2). Removal of promoter DNA upstream of the -35 element also decreased both k(a) and k(2) approximately 10-fold. Upstream DNA extending approximately to base pair -100 was sufficient for maximal association rates of wild-type RNAP with lacUV5 promoter fragments. The alphaCTD and upstream DNA did not affect dissociation rates from the open complex. We suggest that sequence-independent upstream DNA interactions with alphaCTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the alpha-binding sites. In addition to highlighting the functional importance of nonspecific protein-DNA interactions, these results suggest also that UP element-alphaCTD interactions play an even larger role in transcription initiation than appreciated previously.
Ribosomal RNA transcription is the rate-limiting step in ribosome synthesis in bacteria and has been investigated intensely for over half a century. Multiple mechanisms ensure that rRNA synthesis rates are appropriate for the cell's particular growth condition. Recently, important advances have been made in our understanding of rRNA transcription initiation in Escherichia coli. These include (a) a model at the atomic level of the network of protein-DNA and protein-protein interactions that recruit RNA polymerase to rRNA promoters, accounting for their extraordinary strength; (b) discovery of the nonredundant roles of two small molecule effectors, ppGpp and the initiating NTP, in regulation of rRNA transcription initiation; and (c) identification of a new component of the transcription machinery, DksA, that is absolutely required for regulation of rRNA promoter activity. Together, these advances provide clues important for our molecular understanding not only of rRNA transcription, but also of transcription in general.
As an approach to the study of rRNA synthesis in Gram-positive bacteria, we characterized the regulation of the Bacillus subtilis rrnB and rrnO rRNA promoters. We conclude that B. subtilis and Escherichia coli use different strategies to control rRNA synthesis. In contrast to E. coli, it appears that the initiating NTP for transcription from B. subtilis rRNA promoters is GTP, promoter strength is determined primarily by the core promoter (-10/-35 region), and changes in promoter activity always correlate with changes in the intracellular GTP concentration. rRNA promoters in B. subtilis appear to be regulated by changes in the initiating NTP pools, but in some growth transitions, changes in rRNA promoter activity are also dependent on relA, which codes for ppGpp synthetase. In contrast to the situation for E. coli where ppGpp decreases rRNA promoter activity by directly inhibiting RNA polymerase, it appears that ppGpp may not inhibit B. subtilis RNA polymerase directly. Rather, increases in the ppGpp concentration might reduce the available GTP pools, thereby modulating rRNA promoter activity indirectly.
Ribosomal RNA (rRNA) transcription is regulated primarily at the level of initiation from rRNA promoters. The unusual kinetic properties of these promoters result in their specific regulation by two small molecule signals, ppGpp and the initiating NTP, that bind to RNA polymerase (RNAP) at all promoters. We show here that DksA, a protein previously unsuspected as a transcription factor, is absolutely required for rRNA regulation. In deltadksA mutants, rRNA promoters are unresponsive to changes in amino acid availability, growth rate, or growth phase. In vitro, DksA binds to RNAP, reduces open complex lifetime, inhibits rRNA promoter activity, and amplifies effects of ppGpp and the initiating NTP on rRNA transcription, explaining the dksA requirement in vivo. These results expand our molecular understanding of rRNA transcription regulation, may explain previously described pleiotropic effects of dksA, and illustrate how transcription factors that do not bind DNA can nevertheless potentiate RNAP for regulation.
In bacteria, genes are often expressed from multiple promoters to allow for a greater spectrum of regulation. Transcription of rRNA genes in Escherichia coli uses two promoters, rrn P1 and rrn P2. Under the conditions examined previously, the P1 and P2 promoters were regulated in response to many of the same changes in nutritional conditions. We report here that rrn P2 promoters play unique roles in rRNA expression during transitional situations. rrn P2 promoters play a dominant role in rRNA synthesis as cells enter into and persist in stationary phase. rrn P2 promoters also play a role in the rapid increases in rRNA synthesis that occur during outgrowth from stationary phase and during the initial stages of rapid shifts to richer media. We demonstrate that rrnB P2 directly senses the concentrations of guanosine 5'-disphosphate 3'-diphosphate (ppGpp) and the initiating nucleoside triphosphate (iNTP), thereby accounting, at least in part, for the observed patterns of regulation. Our work significantly extends previous information about the regulators responsible for control of the rrn P2 promoters and the relationship between the tandem rRNA promoters.
Previous studies showed that adenosine triphosphate (ATP) concentrations in Escherichia coli changed during certain growth transitions and directly controlled the rate of rRNA transcription initiation at those times. The relationship between ATP concentration and rRNA transcription during steady-state growth is less clear, however. This is because two commonly employed methods for measuring ATP concentrations in bacteria, both of which rely on physical extraction followed by chromatographic separation of small molecules, resulted in dramatically different conclusions about whether ATP concentration changed with steady-state growth rate. Extraction with formic acid indicated that ATP concentration did not change with growth rate, whereas formaldehyde treatment followed by extraction with alkali indicated that ATP concentration increased proportionally to the growth rate. To resolve this discrepancy, we developed a bioassay for ATP based on the expression of a variant of the firefly luciferase enzyme in vivo and measurement of luminescence in cells growing in different conditions. We found that the available ATP concentration did not vary with growth rate, either in wild-type cells or in cells lacking guanosine 5'-diphosphate, 3'-diphosphate, providing insight into the regulation of rRNA transcription. More broadly, the luciferase bioassay described here provides a general method for evaluating the ATP concentration available for biochemical processes in E. coli and potentially in other organisms.
No abstract available.
The mechanosensitive (MS) channels MscS and MscL are essential for the survival of hypoosmotic shock by Escherichia coli cells. We demonstrate that MscS and MscL are induced by osmotic stress and by entry into stationary phase. Reduced levels of MS proteins and reduced expression of mscL- and mscS-LacZ fusions in an rpoS mutant strain suggested that the RNA polymerase holoenzyme containing sigmaS is responsible, at least in part, for regulating production of MS channel proteins. Consistent with the model that the effect of sigmaS is direct, the MscS and MscL promoters both use RNA polymerase containing sigmaS in vitro. Conversely, clpP or rssB mutations, which cause enhanced levels of sigmaS, show increased MS channel protein synthesis. RpoS null mutants are sensitive to hypoosmotic shock upon entry into stationary phase. These data suggest that MscS and MscL are components of the RpoS regulon and play an important role in ensuring structural integrity in stationary phase bacteria.
Early screens for conditional lethal mutations that affected rRNA expression in Escherichia coli identified temperature-sensitive fda mutants (fda encodes the glycolytic enzyme fructose-1,6-diphosphate aldolase). It was shown that these fda(Ts) mutants were severely impaired in rRNA synthesis upon shift to the restrictive temperature, although the mechanism of inhibition was never determined. Here, we bring resolution to this long-standing question by showing that changes in the concentrations of guanosine 5'-diphosphate 3'-diphosphate and initiating nucleoside triphosphates can account for the previously observed effects of fda mutations on rRNA transcription.
rRNA synthesis is the rate-limiting step in ribosome synthesis in Escherichia coli. Its regulation has been described in terms of a negative-feedback control loop in which rRNA promoter activity responds to the amount of translation. The feedback nature of this control system was demonstrated previously by artificially changing ribosome synthesis rates and observing responses of rRNA promoters. However, it has not been demonstrated previously that the initiating nucleoside triphosphate (iNTP) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp), the molecular effectors responsible for controlling rRNA promoters in response to changes in the nutritional environment, are responsible for altering rRNA promoter activities under these feedback conditions. Here, we show that most feedback situations result in changes in the concentrations of both the iNTP and ppGpp and that the directions of these changes are consistent with a role for these two small-molecule regulators in feedback control of rRNA synthesis. In contrast, we observed no change in the level of DNA supercoiling under the feedback conditions examined.
The control of ribosomal RNA transcription is one of the most enduring issues in molecular microbiology, having been subjected to intense scrutiny for over 50 years. Rapid changes in rRNA expression occur during transitions in the bacterial growth cycle and following nutritional shifts during exponential growth. Genetic approaches and measurements of initiating nucleoside triphosphate (iNTP) and guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) concentrations and of rRNA promoter activities showed that rapid changes in the concentrations of iNTPs and ppGpp account for the rapid changes in rRNA expression. The two regulatory signals are nonredundant: changes in iNTP concentration dominate regulation during outgrowth from stationary phase, whereas changes in ppGpp concentration are responsible for regulation following upshifts and downshifts during exponential phase. The results suggest a molecular logic for the use of two homeostatic regulatory mechanisms to monitor different aspects of ribosome activity and provide general insights into the nature of overlapping regulatory circuits.
The C-terminal domain of the Escherichia coli RNA polymerase (RNAP) alpha subunit (alphaCTD) stimulates transcription initiation by interacting with upstream (UP) element DNA and a variety of transcription activators. Here we identify specific substitutions in region 4.2 of sigma 70 (sigma(70)) and in alphaCTD that decrease transcription initiation from promoters containing some, but not all, UP elements. This decrease in transcription derives from a decrease in the initial equilibrium constant for RNAP binding (K(B)). The open complexes formed by the mutant and wild-type RNAPs differ in DNAse I sensitivity at the junction of the alphaCTD and sigma DNA binding sites, correlating with the differences in transcription. A model of the DNA-alphaCTD-sigma region 4.2 ternary complex, constructed from the previously determined X-ray structures of the Thermus aquaticus sigma region 4.2-DNA complex and the E. coli alphaCTD-DNA complex, indicates that the residues identified by mutation in sigma region 4.2 and in alphaCTD are in very close proximity. Our results strongly suggest that alphaCTD, when bound to an UP element proximal subsite, contacts the RNAP sigma(70) subunit, increasing transcription. Previous data from the literature suggest that this same sigma-alphaCTD interaction also plays a role in transcription factor-mediated activation.
The control of ribosome synthesis has been a major focus in molecular biology for over 50 years. As protein synthesis is an essential, yet energetically costly, process, all cells (from bacteria to mammals) devote complex regulatory networks to fine-tune the expression of ribosomal RNA (and therefore ribosome synthesis) to the nutritional environment. In Escherichia coli, ribosomal RNA promoters are among the strongest in the cell and are regulated by trans-acting proteins (Fis and H-NS) and small molecules (guanosine 5'-diphosphate 3'-diphosphate and initiating nucleoside triphosphates). Recent work has dissected many of the molecular mechanisms responsible for the strength and regulation of rRNA promoters.
The seven rRNA operons in Escherichia coli each contain two promoters, rrn P1 and rrn P2. Most previous studies have focused on the rrn P1 promoters. Here we report a systematic analysis of the activity and regulation of the rrnB P2 promoter in order to define the intrinsic properties of rrn P2 promoters and to understand better their contributions to rRNA synthesis when they are in their natural setting downstream of rrn P1 promoters. In contrast to the conclusions reached in some previous studies, we find that rrnB P2 is regulated: it displays clear responses to amino acid availability (stringent control), rRNA gene dose (feedback control), and changes in growth rate (growth rate-dependent control). Stringent control of rrnB P2 requires the alarmone ppGpp, but growth rate-dependent control of rrnB P2 does not require ppGpp. The rrnB P2 core promoter sequence (-37 to +7) is sufficient to serve as the target for growth rate-dependent regulation.
Sinorhizobium meliloti, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family. To facilitate our studies of transcription in S. meliloti, we cloned and characterized the gene for the alpha subunit of RNA polymerase (RNAP). S. meliloti rpoA encodes a 336-amino-acid, 37-kDa protein. Sequence analysis of the region surrounding rpoA identified six open reading frames that are found in the conserved gene order secY (SecY)-adk (Adk)-rpsM (S13)-rpsK (S11)-rpoA (alpha)-rplQ (L17) found in the alpha-proteobacteria. In vivo, S. meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E. coli rpoA, demonstrating that S. meliloti alpha supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of E. coli. In vitro, we reconstituted RNAP holoenzyme from S. meliloti alpha and E. coli beta, beta', and sigma subunits. Similar to E. coli RNAP, the hybrid RNAP supported transcription from an E. coli core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation. We obtained similar results using purified RNAP from S. meliloti. Our results demonstrate that S. meliloti alpha functions are conserved in heterologous host E. coli even though the two alpha subunits are only 51% identical. The ability to utilize E. coli as a heterologous system in which to study the regulation of S. meliloti genes could provide an important tool for our understanding and manipulation of these processes.
We showed previously that rrn P1 promoters require unusually high concentrations of the initiating nucleoside triphosphates (ATP or GTP, depending on the promoter) for maximal transcription in vitro. We proposed that this requirement for high initiating NTP concentrations contributes to control of the rrn P1 promoters from the seven Escherichia coli rRNA operons. However, the previous studies did not prove that variation in NTP concentration affects rrn P1 promoter activity directly in vivo. Here, we create conditions in vivo in which ATP and GTP concentrations are altered in opposite directions relative to one another, and we show that transcription from rrn P1 promoters that initiate with either ATP or GTP follows the concentration of the initiating NTP for that promoter. These results demonstrate that the effect of initiating NTP concentration on rrn P1 promoter activity in vivo is direct. As predicted by a model in which homeostatic control of rRNA transcription results, at least in part, from sensing of NTP concentrations by rrn P1 promoters, we show that inhibition of protein synthesis results in an increase in ATP concentration and a corresponding increase in transcription from rrnB P1. We conclude that translation is a major consumer of purine NTPs, and that NTP-sensing by rrn P1 promoters serves as a direct regulatory link between translation and ribosome synthesis.
Alanine scanning of the Escherichia coli RNA polymerase alpha subunit C-terminal domain (alphaCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of alphaCTD affected class I CRP-dependent transcription from the CC(-61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for alphaCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in alphaCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.
The bacterium Vibrio natriegens can double with a generation time of less than 10 min (R. G. Eagon, J. Bacteriol. 83:736-737, 1962), a growth rate that requires an extremely high rate of protein synthesis. We show here that V. natriegens' high potential for protein synthesis results from an increase in ribosome numbers with increasing growth rate, as has been found for other bacteria. We show that V. natriegens contains a large number of rRNA operons, and its rRNA promoters are extremely strong. The V. natriegens rRNA core promoters are at least as active in vitro as Escherichia coli rRNA core promoters with either E. coli RNA polymerase (RNAP) or V. natriegens RNAP, and they are activated by UP elements, as in E. coli. In addition, the E. coli transcription factor Fis activated V. natriegens rrn P1 promoters in vitro. We conclude that the high capacity for ribosome synthesis in V. natriegens results from a high capacity for rRNA transcription, and the high capacity for rRNA transcription results, at least in part, from the same factors that contribute most to high rates of rRNA transcription in E. coli, i.e., high gene dose and strong activation by UP elements and Fis.
Fis is a versatile transactivator that functions at many different promoters. Fis activates transcription at the RpoS-dependent proP P2 promoter when bound to a site that overlaps the minus sign35 hexamer by a mechanism that requires the C-terminal domain of the alpha subunit of RNA polymerase (alphaCTD). The region on Fis responsible for activating transcription through the alphaCTD has been localized to a short beta-turn near the DNA-binding determinant on one subunit of the Fis homodimer. We report here that Fis-dependent activation of proP P2 transcription requires two discrete regions on the alphaCTD. One region, consisting of residues 264-265 and 296-297, mediates DNA binding. A second patch, comprising amino acid residues 271-273, forms a ridge on the surface of the alphaCTD that we propose interacts with Fis. The accompanying paper shows that these same regions on alphaCTD are utilized for transcriptional activation at the rrnB and rrnE P1 promoters by Fis bound to a site upstream of the core promoter (centered at minus sign71/minus sign72). In addition to stimulation of proP P2 transcription by Fis, CRP co-activates this promoter when bound to a remote site upstream from the promoter (centered at -121.5). RNA polymerase preparations lacking one alphaCTD of the alpha dimer were employed to demonstrate that the beta'-associated alpha(II)CTD was utilized preferentially by Fis at proP P2 in the presence and absence of CRP. These experiments define the overall architecture of the proP P2 initiation complex where Fis and CRP each function through a different alphaCTD.
The transcription factor Fis activates the Escherichia coli rRNA promoters rrnB P1 and rrnE P1 by binding to sites centered at -71 and -72, respectively, and interacting with the C-terminal domain of the alpha subunit of RNA polymerase (RNAP alphaCTD). To understand the mechanism of activation by Fis at these promoters, we used oriented alpha-heterodimeric RNAPs and heterodimers of Fis to determine whether one or both subunits of alpha and Fis participate in the alphaCTD-Fis interaction. Our results imply that only one alphaCTD in the alpha dimer and only one activation-proficient subunit in the Fis dimer are required for activation by Fis. A library of alanine substitutions in alpha was used to identify the alphaCTD determinants required for Fis-dependent transcription at rrnB P1 and rrnE P1. We propose that the transcriptional activation region of the promoter-proximal subunit of the Fis dimer interacts with a determinant that includes E273 of one alphaCTD to activate transcription. We further suggest that the Fis contact to alphaCTD results in alphaCTD interactions with DNA that differ somewhat from those that occur at UP elements in the absence of Fis. The accompanying paper shows that the 273 determinant on alphaCTD is also targeted by Fis at the proP P2 promoter where the activator binds overlapping the -35 hexamer. Thus, similar Fis-alphaCTD interactions are used for activation of transcription when the activator is bound at very different positions on the DNA.
Although more than 30 Escherichia coli promoters utilize the RNA polymerase holoenzyme containing sigmaS (EsigmaS), and it is known that there is some overlap between the promoters recognized by EsigmaS and by the major E. coli holoenzyme (Esigma70), the sequence elements responsible for promoter recognition by EsigmaS are not well understood. To define the DNA sequences recognized best by EsigmaS in vitro, we started with random DNA and enriched for EsigmaS promoter sequences by multiple cycles of binding and selection. Surprisingly, the sequences selected by EsigmaS contained the known consensus elements (-10 and -35 hexamers) for recognition by Esigma70. Using genetic and biochemical approaches, we show that EsigmaS and Esigma70 do not achieve specificity through 'best fit' to different consensus promoter hexamers, the way that other forms of holoenzyme limit transcription to discrete sets of promoters. Rather, we suggest that EsigmaS-specific promoters have sequences that differ significantly from the consensus in at least one of the recognition hexamers, and that promoter discrimination against Esigma70 is achieved, at least in part, by the two enzymes tolerating different deviations from consensus. DNA recognition by EsigmaS versus Esigma70 thus presents an alternative solution to the problem of promoter selectivity.
We have previously shown that the activity of the Escherichia coli rRNA promoter rrnB P1 in vitro depends on the concentration of the initiating nucleotide, ATP, and can respond to changes in ATP pools in vivo. We have proposed that this nucleoside triphosphate (NTP) sensing might contribute to regulation of rRNA transcription. To test this model, we have measured the ATP requirements for transcription from 11 different rrnB P1 core promoter mutants in vitro and compared them with the regulatory responses of the same promoters in vivo. The seven rrnB P1 variants that required much lower ATP concentrations than the wild-type promoter for efficient transcription in vitro were defective for response to growth rate changes in vivo (growth rate-dependent regulation). In contrast, the four variants requiring high ATP concentrations in vitro (like the wild-type promoter) were regulated with the growth rate in vivo. We also observed a correlation between NTP sensing in vitro and the response of the promoters in vivo to deletion of the fis gene (an example of homeostatic control), although this relationship was not as tight as for growth rate-dependent regulation. We conclude that the kinetic features responsible for the high ATP concentration dependence of the rrnB P1 promoter in vitro are responsible, at least in part, for the promoter's regulation in vivo, consistent with the model in which rrnB P1 promoter activity can be regulated by changes in NTP pools in vivo (or by hypothetical factors that work at the same kinetic steps that make the promoter sensitive to NTPs).
The high activity of the rrnB P1 promoter in Escherichia coli results from a cis-acting DNA sequence, the UP element, and a trans-acting transcription factor, FIS. In this study, we examine the effects of FIS and the UP element at the other six rrn P1 promoters. We find that UP elements are present at all of the rrn P1 promoters, but they make different relative contributions to promoter activity. Similarly, FIS binds upstream of, and activates, all seven rrn P1 promoters but to different extents. The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each rrn P1 promoter. Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most rrn P1 promoters compared to rrnB P1. Our studies indicate that the overall activities of the seven rrn P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter. These studies have implications for the control of gene expression of unlinked multigene families.
The UP element stimulates transcription from the rrnB P1 promoter through a direct interaction with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). We investigated the effect on transcription from rrnB P1 of varying both the location of the UP element and the length of the alpha subunit interdomain linker, separately and in combination. Displacement of the UP element by a single turn of the DNA helix resulted in a large decrease in transcription from rrnB P1, while displacement by half a turn or two turns totally abolished UP element-dependent transcription. Deletions of six or more amino acids from within the alpha subunit linker resulted in a decrease in UP element-dependent stimulation, which correlated with decreased binding of alphaCTD to the UP element. Increasing the alpha linker length was less deleterious to RNA polymerase function at rrnB P1 but did not compensate for the decrease in activation that resulted from displacing the UP element. Our results suggest that the location of the UP element at rrnB P1 is crucial to its function and that the natural length of the alpha subunit linker is optimal for utilisation of the UP element at this promoter.
The alpha subunit of E. coli RNAP plays an important role in the recognition of many promoters by binding to the A+T-rich UP element, a DNA sequence located upstream of the recognition elements for the sigma subunit, the -35 and -10 hexamers. We examined DNA-RNAP interactions using high resolution interference and protection footprinting methods and using the minor groove-binding drug distamycin. Our results suggest that alpha interacts with bases in the DNA minor groove and with the DNA backbone along the minor groove, but that UP element major groove surfaces do not make a significant contribution to alpha binding. On the basis of these and previous results, we propose a model in which alpha contacts UP element DNA through amino acid residues located in a pair of helix-hairpin-helix motifs. Furthermore, our experiments extend existing information about recognition of the core promoter by sigma(70) by identifying functional groups in the major grooves of the -35 and -10 hexamers in which modifications interfere with RNAP binding. These studies greatly improve the resolution of our picture of the promoter-RNAP interaction.
Strains containing ppGpp, a nucleotide whose synthesis is dependent on the RelA and SpoT proteins of Escherichia coli, display slightly lower rRNA promoter activity and much higher amino acid biosynthesis/transport promoter activity than deltarelAdeltaspoT strains. In the accompanying paper, we show that ppGpp directly inhibits rRNA promoter activity in vitro by decreasing the lifetime of the rrn P1 open complex. However, ppGpp does not stimulate amino acid promoter activity in vitro. We show here that RNA polymerase (RNAP) mutants, selected to confer prototrophy to deltarelAdeltaspoT strains, mimic the effects of ppGpp on wild-type RNAP. Based on the positions of the mutant residues that confer prototrophy in the structure of core RNAP, we suggest molecular models for how the mutants, and by analogy ppGpp, generally decrease the lifetime of open complexes. We show that amino acid promoters require higher concentrations of RNAP for function in vitro and in vivo than control promoters, and are more sensitive to competition for RNAP in vivo than control promoters. Furthermore, we show that the requirement of an amino acid promoter for ppGpp in vivo can be alleviated by increasing its rate-limiting RNAP-binding step. Our data are consistent with a previously proposed passive model in which ppGpp inhibits stable RNA synthesis directly by reducing the lifetime of the rrn P1 open complex, liberating enough RNAP to stimulate transcription from amino acid promoters. Our data also place considerable constraints on models invoking hypothetical factors that might increase amino acid promoter activity in a ppGpp-dependent fashion.
To determine the role of ppGpp in both negative and positive regulation of transcription initiation during exponential growth in Escherichia coli, we examined transcription in vivo and in vitro from the growth-rate-dependent rRNA promoter rrnB P1 and from the inversely growth-rate-dependent amino acid biosynthesis/transport promoters PargI, PhisG, PlysC, PpheA, PthrABC, and PlivJ. rrnB P1 promoter activity was slightly higher at all growth-rates in strains unable to synthesize ppGpp (deltarelAdeltaspoT) than in wild-type strains. Consistent with this observation and with the large decrease in rRNA transcription during the stringent response (when ppGpp levels are much higher), ppGpp inhibited transcription from rrnB P1 in vitro. In contrast, amino acid promoter activity was considerably lower in deltarelAdeltaspoT strains than in wild-type strains, but ppGpp had no effect on amino acid promoter activity in vitro. Detailed kinetic analysis in vitro indicated that open complexes at amino acid promoters formed much more slowly and were much longer-lived than rrnB P1 open complexes. ppGpp did not increase the rates of association with, or escape from, amino acid promoters in vitro, consistent with its failure to stimulate transcription directly. In contrast, ppGpp decreased the half-lives of open complexes at all promoters, whether the half-life was seconds (rrnB P1) or hours (amino acid promoters). The results described here and in the accompanying paper indicate that ppGpp directly inhibits transcription, but only from promoters like rrnB P1 that make short-lived open complexes. The results indicate that stimulation of amino acid promoters occurs indirectly. The accompanying paper evaluates potential models for positive control of amino acid promoters by ppGpp that might explain the requirement of ppGpp for amino acid prototrophy.
In recent years, it has become clear that promoter recognition by bacterial RNA polymerase involves interactions not only between core promoter elements and the sigma subunit, but also between a DNA element upstream of the core promoter and the alpha subunit. DNA binding by alpha can increase transcription dramatically. Here we review the current state of our understanding of the alpha interaction with DNA during basal transcription initiation (i.e. in the absence of proteins other than RNA polymerase) and activated transcription initiation (i.e. when stimulated by transcription factors).
We recently identified Escherichia coli RNA polymerase (RNAP) mutants (RNAP beta' Delta215-220 and beta RH454) that form extremely unstable complexes with rRNA P1 (rrn P1) core promoters. The mutant RNAPs reduce transcription and alter growth rate-dependent regulation of rrn P1 core promoters, because the mutant RNAPs require higher concentrations of the initiating nucleoside triphosphate (NTP) for efficient transcription from these promoters than are present in vivo. Nevertheless, the mutants grow almost as well as wild-type cells, suggesting that rRNA synthesis is not greatly perturbed. We report here that the rrn transcription factor FIS activates the mutant RNAPs more strongly than wild-type RNAP, thereby compensating for the altered properties of the mutant RNAPs. FIS activates the mutant RNAPs, at least in part, by reducing the apparent K(ATP) for the initiating NTP. This and other results suggest that FIS affects a step in transcription initiation after closed-complex formation in addition to its stimulatory effect on initial RNAP binding. FIS and NTP levels increase with growth rate, suggesting that changing FIS concentrations, in conjunction with changing NTP concentrations, are responsible for growth rate-dependent regulation of rrn P1 transcription in the mutant strains. These results provide a dramatic demonstration of the interplay between regulatory mechanisms in rRNA transcription.
ProP is an integral membrane transporter of proline, glycine betaine, and several other osmoprotecting compounds. Fis plus RpoS collaborate to promote a burst of proP transcription in late exponential growth phase. This brief period of ProP synthesis enables stationary phase cells to cope with a potential hyperosmotic shock. Fis activates the RpoS (sigma(38))-dependent proP P2 promoter by binding to a site within the promoter region centered at -41 and thus functions as a class II activator. We show here that activation by Fis at this promoter is completely dependent upon the alpha-CTD of RNA polymerase and that the activation domain on Fis is localized to a four amino acid ridge on the surface of Fis adjacent to the helix-turn-helix DNA binding domain in only one subunit of the homodimer. Fis mutants containing amino acid substitutions within this region are defective in cooperative binding interactions with the sigma(38)-form of RNA polymerase. Some of these substitutions also alter interactions with DNA sequences flanking the core binding site, but we show that changes in Fis-mediated curvature do not affect promoter activity. We conclude that the same amino acids are used by Fis to activate transcription from a class I (-71, rrnB P1) and class II (-41, proP P2) location, but this region is distinct from that required to regulate the Hin site-specific DNA inversion reaction.
We demonstrate here that the previously described bacterial promoter upstream element (UP element) consists of two distinct subsites, each of which, by itself, can bind the RNA polymerase holoenzyme alpha subunit carboxy-terminal domain (RNAP alphaCTD) and stimulate transcription. Using binding-site-selection experiments, we identify the consensus sequence for each subsite. The selected proximal subsites (positions -46 to -38; consensus 5'-AAAAAARNR-3') stimulate transcription up to 170-fold, and the selected distal subsites (positions -57 to -47; consensus 5'-AWWWWWTTTTT-3') stimulate transcription up to 16-fold. RNAP has subunit composition alpha(2)betabeta'sigma and thus contains two copies of alphaCTD. Experiments with RNAP derivatives containing only one copy of alphaCTD indicate, in contrast to a previous report, that the two alphaCTDs function interchangeably with respect to UP element recognition. Furthermore, function of the consensus proximal subsite requires only one copy of alphaCTD, whereas function of the consensus distal subsite requires both copies of alphaCTD. We propose that each subsite constitutes a binding site for a copy of alphaCTD, and that binding of an alphaCTD to the proximal subsite region (through specific interactions with a consensus proximal subsite or through nonspecific interactions with a nonconsensus proximal subsite) is a prerequisite for binding of the other alphaCTD to the distal subsite.
When the number of rRNA (rrn) operons in an Escherichia coli cells is increased by adding an rrn operon on a multicopy plasmid, the rate of rRNA expression per operon is reduced to maintain a constant concentration of rRNA in the cell. We have used electron microscopy to examine rRNA transcription in cells containing a multicopy plasmid carrying rrnB. We found that there were fewer RNA polymerase molecules transcribing the rrn genes, as predicted from previous gene dosage studies. Furthermore, RNA polymerase molecules were arranged in irregularly spaced groups along the operon. No apparent pause or transcription termination sites that would account for the irregular spacing of the groups of polymerase molecules were observed. We also found that the overall transcription elongation rate was unchanged when the rrn gene dosage was increased. Our data suggest that when rrn gene dosage is increased, initiation events, or promoter-proximal elongation events, are interrupted at irregular time intervals.
No abstract available.
The transcription factor FIS has been implicated in the regulation of several stable RNA promoters, including that for the major tRNALeu species in Escherichia coli, tRNA1Leu. However, no evidence for direct involvement of FIS in tRNA1Leu expression has been reported. We show here that FIS binds to a site upstream of the leuV promoter (centered at -71) and that it directly stimulates leuV transcription in vitro. A mutation in the FIS binding site reduces transcription from a leuV promoter in strains containing FIS but has no effect on transcription in strains lacking FIS, indicating that FIS contributes to leuV expression in vivo. We also find that RNA polymerase forms an unusual heparin-sensitive complex with the leuV promoter, having a downstream protection boundary of approximately -7, and that the first two nucleotides of the transcript, GTP and UTP, are required for formation of a heparin-stable complex that extends downstream of the transcription start site. These studies have implications for the regulation of leuV transcription.
CooA, a member of the cAMP receptor protein (CRP) family, is a CO-sensing transcription activator from Rhodospirillum rubrum that binds specific DNA sequences in response to CO. The location of the CooA-binding sites relative to the start sites of transcription suggested that the CooA-dependent promoters are analogous to class II CRP-dependent promoters. In this study, we developed an in vivo CooA reporter system in Escherichia coli and an in vitro transcription assay using RNA polymerases (RNAP) from E. coli and from Rhodobacter sphaeroides to study the transcription properties of CooA and the protein-protein interaction between CooA and RNAP. The ability of CooA to activate CO-dependent transcription in vivo in heterologous backgrounds suggested that CooA is sufficient to direct RNAP to initiate transcription and that no other factors are required. This hypothesis was confirmed in vitro with purified CooA and purified RNAP. Use of a mutant form of E. coli RNAP with alpha subunits lacking their C-terminal domain (alpha-CTD) dramatically decreased CooA-dependent transcription of the CooA-regulated R. rubrum promoter PcooF in vitro, which indicates that alpha-CTD plays an important role in this activation. DNase I footprinting analysis showed that CooA facilitates binding of wild-type RNAP, but not alpha-CTD-truncated RNAP, to PcooF. This facilitated binding provides evidence for a direct contact between CooA and alpha-CTD of RNAP during activation of transcription. Mapping the CooA-contact site in alpha-CTD suggests that CooA is similar but not identical to CRP in terms of its contact sites to the alpha-CTD at class II promoters.
We screened a collection of single alanine residue substitution mutants spanning the entire C-terminal domain of the alpha subunit (alphaCTD) of Escherichia coli RNA polymerase (RNAP) for defects in rho-dependent transcription termination at lambdatR1 in vivo and in vitro, and thereby identified a patch of amino acid residues in the alphaCTD required for efficient rho-dependent termination. NusA addition led to the stimulation of rho-dependent termination under our conditions in vitro. The termination defects of a few mutant RNAPs could be attributed to altered interactions with the NusA protein, but rho-dependent termination by most of the defective RNAPs was still stimulated normally by NusA. The NusA-enhanced transcription pausing behaviors of the mutant RNAPs did not always correlate with their rho-dependent termination phenotypes. We conclude that the alphaCTD is a target for interactions with NusA that influence both termination and pausing, but in addition it participates in rho-dependent transcription termination in a NusA-independent manner.
Upstream A-tracts stimulate transcription from a variety of bacterial promoters, and this has been widely attributed to direct effects of the intrinsic curvature of A-tract-containing DNA. In this work we report experiments that suggest a different mechanism for the effects of upstream A-tracts on transcription. The similarity of A-tract-containing sequences to the adenine- and thymine-rich upstream recognition elements (UP elements) found in some bacterial promoters suggested that A-tracts might increase promoter activity by interacting with the alpha subunit of RNA polymerase (RNAP). We found that an A-tract-containing sequence placed upstream of the Escherichia coli lac or rrnB P1 promoters stimulated transcription both in vivo and in vitro, and that this stimulation required the C-terminal (DNA-binding) domain of the RNAP alpha subunit. The A-tract sequence was protected by wild-type RNAP but not by alpha-mutant RNAPs in footprints. The effect of the A-tracts on transcription was not as great as that of the most active UP elements, consistent with the degree of similarity of the A-tract sequence to the UP element consensus. A-tracts functioned best when positioned close to the -35 hexamer rather than one helical turn farther upstream, similar to the positioning optimal for UP element function. We conclude that A-tracts function as UP elements, stimulating transcription by providing binding site(s) for the RNAP alphaCTD, and we suggest that these interactions could contribute to the previously described wrapping of promoter DNA around RNAP.
The alpha subunit of Escherichia coli RNA polymerase (RNAP) participates in promoter recognition through specific interactions with UP element DNA, a region upstream of the recognition hexamers for the sigma subunit (the -10 and -35 hexamers). UP elements have been described in only a small number of promoters, including the rRNA promoter rrnB P1, where the sequence has a very large (30- to 70-fold) effect on promoter activity. Here, we analyzed the effects of upstream sequences from several additional E. coli promoters (rrnD P1, rrnB P2, lambda pR, lac, merT, and RNA II). The relative effects of different upstream sequences were compared in the context of their own core promoters or as hybrids to the lac core promoter. Different upstream sequences had different effects, increasing transcription from 1.5- to approximately 90-fold, and several had the properties of UP elements: they increased transcription in vitro in the absence of accessory protein factors, and transcription stimulation required the C-terminal domain of the RNAP alpha subunit. The effects of the upstream sequences correlated generally with their degree of similarity to an UP element consensus sequence derived previously. Protection of upstream sequences by RNAP in footprinting experiments occurred in all cases and was thus not a reliable indicator of UP element strength. These data support a modular view of bacterial promoters in which activity reflects the composite effects of RNAP interactions with appropriately spaced recognition elements (-10, -35, and UP elements), each of which contributes to activity depending on its similarity to the consensus.
The UP element, a component of bacterial promoters located upstream of the -35 hexamer, increases transcription by interacting with the RNA polymerase alpha-subunit. By using a modification of the SELEX procedure for identification of protein-binding sites, we selected in vitro and subsequently screened in vivo for sequences that greatly increased promoter activity when situated upstream of the Escherichia coli rrnB P1 core promoter. A set of 31 of these upstream sequences increased transcription from 136- to 326-fold in vivo, considerably more than the natural rrnB P1 UP element, and was used to derive a consensus sequence: -59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn -38. The most active selected sequence contained the derived consensus, displayed all of the properties of an UP element, and the interaction of this sequence with the alpha C-terminal domain was similar to that of previously characterized UP elements. The identification of the UP element consensus should facilitate a detailed understanding of the alpha-DNA interaction. Based on the evolutionary conservation of the residues in alpha responsible for interaction with UP elements, we suggest that the UP element consensus sequence should be applicable throughout eubacteria, should generally facilitate promoter prediction, and may be of use for biotechnological applications.
Mutations in Escherichia coli rpoB or rpoC, selected for the ability to confer prototrophy on relA spoT strains, were found to affect transcription from rrn P1 promoters. Two mutant strains (beta RH454 and beta' delta 215-220) reduced transcription of rrn P1 core promoter-lacZ fusions but not of control promoter-lacZ fusions. Purified mutant RNAPs formed complexes with rrn P1 promoters that were much less stable than those formed by wild-type RNAP and required high concentrations of the initiating NTP for efficient rrn P1 transcription. The instability of the rrn P1 core promoter complexes with the mutant RNAPs and their altered regulatory properties support a recently proposed model for the control of rRNA transcription by changing concentrations of the initiating NTPs. We further suggest that destabilization of promoter complexes by the mutant RNAPs mimics effects of ppGpp, decreasing or increasing transcription depending on the kinetic properties of the specific promoter.
Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRP-dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase alpha subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within alphaCTD for transcription activation at a Class II CRP-dependent promoter. Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285-288 and 317. Residues 285-288 and 317 comprise a discrete 20x10 A surface on alphaCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between alpha subunits and CRP, but do not affect DNA binding by alpha subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in alphaCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for alphaCTD-CRP interaction in Class I CRP-dependent transcription.
We have characterized the Chlamydia trachomatis ribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purified C. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential -10 and -35 elements, analogous to Escherichia coli promoters recognized by E-sigma70. We identified a novel AT-rich region immediately downstream of the -35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the -35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.
rRNA transcription in Escherichia coli is activated by the FIS protein, which binds upstream of rrnp1 promoters and interacts directly with RNA polymerase. Analysis of the contribution of FIS to rrn transcription under changing physiological conditions is complicated by several factors: the wide variation in cellular FIS concentrations with growth conditions, the contributions of several other regulatory systems to rRNA synthesis, and the pleiotropy of fis mutations. In this report, we show by in vivo footprinting and Western blot analysis that occupancy of the rrnBp1 FIS sites correlates with cellular levels of FIS. We find, using two methods of measurement (pulse induction of a FIS-activated hybrid promoter and primer extension from an unstable transcript made from rrnBp1), that the extent of transcription activation by FIS parallels the degree of FIS site occupancy and therefore cellular FIS levels. FIS activates transcription throughout exponential growth at low culture density, but rrnp1 transcription increases independently of FIS immediately following upshift, before FIS accumulates. These results support the model that FIS is one of a set of overlapping signals that together contribute to transcription from rrnp1 promoters during steady-state growth.
The sequence of a promoter determines not only the efficiency with which it forms a complex with RNA polymerase, but also the concentration of nucleoside triphosphate (NTP) required for initiating transcription. Escherichia coli ribosomal RNA (rrn P1) promoters require high initiating NTP concentrations for efficient transcription because they form unusually short-lived complexes with RNA polymerase; high initiating NTP concentrations [adenosine or guanosine triphosphate (ATP or GTP), depending on the rrn P1 promoter] are needed to bind to and stabilize the open complex. ATP and GTP concentrations, and therefore rrn P1 promoter activity, increase with growth rate. Because ribosomal RNA transcription determines the rate of ribosome synthesis, the control of ribosomal RNA transcription by NTP concentration provides a molecular explanation for the growth rate-dependent control and homeostatic regulation of ribosome synthesis.
The synthesis of ribosomal RNA is the rate-limiting step in ribosome synthesis in bacteria. There are multiple mechanisms that determine the rate of rRNA synthesis. Ribosomal RNA promoter sequences have evolved for exceptional strength and for regulation in response to nutritional conditions and amino acid availability. Strength derives in part from an extended RNA polymerase (RNAP) recognition region involving at least two RNAP subunits, in part from activation by a transcription factor and in part from modification of the transcript by a system that prevents premature termination. Regulation derives from at least two mechanistically distinct systems, growth rate-dependent control and stringent control. The mechanisms contributing to rRNA transcription work together and compensate for one another when individual systems are rendered inoperative.
FIS, a site-specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli. The ribosomal RNA promoter rrnB P1 is activated 3- to 7-fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C-terminal domain (CTD) of the alpha subunit of RNA polymerase (RNAP). In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1. These side chains map to positions 68, 71 and 74, in and flanking a surface-exposed loop adjacent to the helix-turn-helix DNA binding motif of the protein. We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP alphaCTD. Our results suggest a model for FIS-mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP alphaCTD near the DNA surface. Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP alphaCTD, suggesting that this is a common architecture at bacterial promoters.
Integration host factor (IHF) can activate transcription from the early promoter (Pe) of bacteriophage Mu both directly and indirectly. Indirect activation occurs through alleviation of H-NS-mediated repression of the Pe promoter (P. Van Ulsen, M. Hillebrand, L. Zulianello, P. Van de Putte, and N. Goosen, Mol. Microbiol. 21:567-578, 1996). The direct activation involves the C-terminal domain of the alpha subunit (alphaCTD) of RNA polymerase. We investigated which residues in the alphaCTD are important for IHF-mediated activation of the Pe promoter. Initial in vivo screening, using a set of substitution mutants derived from an alanine scan (T. Gaal, W. Ross, E. E. Blatter, T. Tang, X. Jia, V. V. Krishnan, N. Assa-Munt, R. Ebright, and R. L. Gourse, Genes Dev. 10:16-26, 1996; H. Tang, K. Severinov, A. Goldfarb, D. Fenyo, B. Chait, and R. H. Ebright, Genes Dev. 8:3058-3067, 1994), indicated that the residues, which are required for transcription activation by the UP element of the rrnB P1 promoter (T. Gaal, W. Ross, E. E. Blatter, T. Tang, X. Jia, V. V. Krishnan, N. Assa-Munt, R. Ebright, and R. L. Gourse, Genes Dev. 10:16-26, 1996), are also important for Pe expression in the presence of IHF. Two of the RNA polymerase mutants, alphaR265A and alphaG296A, that affected Pe expression most in vivo were subsequently tested in in vitro transcription experiments. Mutant RNA polymerase with alphaR265A showed no IHF-mediated activation and a severely reduced basal level of transcription from the Pe promoter. Mutant RNA polymerase with alphaG296A resulted in a slightly reduced transcription from the Pe promoter in the absence of IHF but could still be activated by IHF. These results indicate that interaction of the alphaCTD with DNA is involved not only in the IHF-mediated activation of Pe transcription but also in maintaining the basal level of transcription from this promoter. Mutational analysis of the upstream region of the Pe promoter identified a sequence, positioned from -39 to -51 with respect to the transcription start site, that is important for basal Pe expression, presumably through binding of the alphaCTD. The role of the alphaCTD in IHF-mediated stimulation of transcription from the Pe promoter is discussed.
The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have identified mutants of the FIS protein resulting in reduced rrnB P1 transcription activation that nevertheless retain the ability to bind DNA in vivo. The mutations map to amino acid 74, the N-terminal amino acid of the protein's helix-turn-helix DNA binding motif, and to amino acids 71 and 72 in the adjoining surface-exposed loop. In vitro analyses of one of the activation-defective mutants (with a G-to-S mutation at position 72) indicates that it binds to and bends rrnB P1 FIS site I DNA the same as wild-type FIS. These data suggest that amino acids in this region of FIS are required for transcription activation by contacting RNA polymerase directly, independent of any other role(s) this region may play in DNA binding or protein-induced bending.
The Escherichia coli RNA polymerase alpha-subunit binds through its carboxy-terminal domain (alpha CTD) to a recognition element, the upstream (UP) element, in certain promoters. We used genetic and biochemical techniques to identify the residues in alpha CTD important for UP-element-dependent transcription and DNA binding. These residues occur in two regions of alpha CTD, close to but distinct from, residues important for interactions with certain transcription activators. We used NMR spectroscopy to determine the secondary structure of alpha CTD, alpha CTD contains a nonstandard helix followed by four alpha-helices. The two regions of alpha CTD important for DNA binding correspond to the first alpha-helix and the loop between the third and fourth alpha-helices. The alpha CTD DNA-binding domain architecture is unlike any DNA-binding architecture identified to date, and we propose that alpha CTD has a novel mode of interaction with DNA. Our results suggest models for alpha CTD-DNA and alpha CTD-DNA-activator interactions during transcription initiation.
Escherichia coli uses at least two regulatory systems, stringent control and growth-rate-dependent control, to adjust rRNA output to amino acid availability and the steady-state growth rate, respectively. We examined transcription from rrnB P1 promoters containing or lacking the cis-acting UP element and FIS protein binding sites after amino acid starvation. The "core promoter" responds to amino acid starvation like the full-length wild-type promoter; thus, neither the UP element nor FIS plays a role in stringent control. To clarify the relationship between growth-rate-dependent regulation and stringent control, we measured transcription from growth-rate-independent promoters during amino acid starvation. Four rrnB P1 mutants defective for growth-rate control and two other growth-rate-independent promoters (rrnB P2 and pS10) still displayed stringent regulation. Thus, the two systems have different promoter determinants, consistent with the idea that they function by different mechanisms. Two mutations disrupted stringent control of rrnB P1: (i) a multiple base change in the "discriminator" region between the -10 hexamer and the transcription start site and (ii) a double substitution making the promoter resemble the E sigma 70 consensus promoter. These results have important implications for the mechanisms of both stringent control and growth-rate-dependent control of rRNA transcription.
The E. coli rrnB P1 promoter owes its strength, in part, to the transcriptional activator protein FIS. FIS binds to three sites upstream of the RNA polymerase (RNAP) binding site and increases transcription in vivo four to ten-fold. In this report, hydroxyl radical and DMS footprinting analyses show that FIS binds to its three sites along one side of the DNA helix, and that FIS bound at the promoter-proximal site (site I) and RNAP bound at the promoter are in close proximity. The binding of FIS at site I and RNAP at the promoter are mutually cooperative. These observations support a model for direct interaction between the FIS protein bound at site I and RNAP in transcription activation at rrnB P1. We also find that FIS does not bind cooperatively to its three sites upstream of rrnB P1, and that the relatively small activation associated with FIS bound at sites II and III does not result indirectly by facilitation of binding of FIS to site I.
Using limited proteolysis, we show that the Escherichia coli RNA polymerase alpha subunit consists of an N-terminal domain comprised of amino acids 8-241, a C-terminal domain comprised of amino acids 249-329, and an unstructured and/or flexible interdomain linker. We have carried out a detailed structural and functional analysis of an 85 amino acid proteolytic fragment corresponding to the C-terminal domain (alpha CTD-2). Our results establish that alpha CTD-2 has a defined secondary structure (approximately 40% alpha helix, approximately 0% beta sheet). Our results further establish that alpha CTD-2 is a dimer and that alpha CTD-2 exhibits sequence-specific DNA binding activity. Our results suggest a model for the mechanism of involvement of alpha in transcription activation by promoter upstream elements and upstream-binding activator proteins.
We have extended our previous studies of the DNA sequences required for growth rate-dependent control of rRNA transcription in Escherichia coli. Utilizing a reporter system suitable for evaluation of promoters with low activities, we have found that the core promoter region of rrnB P1 (-41 to +1 with respect to the transcription initiation site) is sufficient for growth rate-dependent control of transcription, both in the presence and in the absence of guanosine 3'-diphosphate 5'-diphosphate (ppGpp). The core promoter contains the -10 and -35 hexamers for recognition by the sigma 70 subunit of RNA polymerase but lacks the upstream (UP) element, which increases transcription by interacting with the alpha subunit of RNA polymerase. It also lacks the binding sites for the positive transcription factor FIS. Thus, the UP element, FIS, and ppGpp are not needed for growth rate-dependent regulation of rRNA transcription. In addition, we find that several core promoter mutations, including -10 and -35 hexamer substitutions, severely reduce rrnB P1 activity without affecting growth rate-dependent control. Thus, a high activity is not a determinant of growth rate regulation of rRNA transcription.
DNA sequences upstream of the rrnB P1 core promoter (-10, -35 region) increase transcription more than 300-fold in vivo and in vitro. This stimulation results from a cis-acting DNA sequence, the UP element, which interacts directly with the alpha subunit of RNA polymerase, increasing transcription about 30-fold, and from a positively acting transcription factor, FIS, which increases expression another 10-fold. A DNA region exhibiting a high degree of intrinsic curvature has been observed upstream of the rrnB P1 core promoter and has thus been often cited as an example of the effect of bending on transcription. However, the precise position of the curvature has not been determined. We address here whether this bend is in fact related to activation of rRNA transcription. Electrophoretic analyses were used to localize the major bend in the rrnB P1 upstream region to position approximately -100 with respect to the transcription initiation site. Since most of the effect of upstream sequences on transcription results from DNA between the -35 hexamer and position -88, i.e. downstream of the bend center, these studies indicate that the curvature leading to the unusual electrophoretic behavior of the upstream region does not play a major role in activation of rRNA transcription. Minor deviations from normal electrophoretic behavior were associated with the region just upstream of the -35 hexamer and could conceivably influence interactions between the UP element and the alpha subunit of RNA polymerase.
The extraordinary strength of the Escherichia coli rRNA promoter rrnB P1 derives primarily from sequences upstream of the core (-10, -35) region. We find that sequences between -40 and -60 increase the activity of this promoter at least 30-fold in vitro and in vivo. This region, which we refer to as the upstream (UP) element, is located between the -35 consensus hexamer and the previously characterized binding sites for the rRNA transcription factor Fis. The effect of the UP element is independent of Fis in vivo, and independent of any other proteins besides RNA polymerase (RNAP) in vitro. The UP element increases the overall second-order rate constant for association of RNAP with the promoter (ka) and probably the apparent overall first-order isomerization constant (ki). Together with the previously reported protection of the UP element region by RNAP in footprinting experiments, these results indicate that rrnB P1 has an "extended" promoter structure, consisting of the UP element and the core promoter region. We find that the UP element is a separable promoter module that can function to increase the activity of the lac core promoter in an rrnB P1-lac hybrid promoter construct. A functional UP element is not absolutely essential for stimulation of rrnB P1 by the Fis protein.
A DNA sequence rich in (A+T), located upstream of the -10, -35 region of the Escherichia coli ribosomal RNA promoter rrnB P1 and called the UP element, stimulates transcription by a factor of 30 in vivo, as well as in vitro in the absence of protein factors other than RNA polymerase (RNAP). When fused to other promoters, such as lacUV5, the UP element also stimulates transcription, indicating that it is a separate promoter module. Mutations in the carboxyl-terminal region of the alpha subunit of RNAP prevent stimulation of these promoters by the UP element although the mutant enzymes are effective in transcribing the "core" promoters (those lacking the UP element). Protection of UP element DNA by the mutant RNAPs is severely reduced in footprinting experiments, suggesting that the selective decrease in transcription might result from defective interactions between alpha and the UP element. Purified alpha binds specifically to the UP element, confirming that alpha acts directly in promoter recognition. Transcription of three other promoters was also reduced by the COOH-terminal alpha mutations. These results suggest that UP elements comprise a third promoter recognition region (in addition to the -10, -35 recognition hexamers, which interact with the sigma subunit) and may account for the presence of (A+T)-rich DNA upstream of many prokaryotic promoters. Since the same alpha mutations also block activation by some transcription factors, mechanisms of promoter stimulation by upstream DNA elements and positive control by certain transcription factors may be related.
The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Res. 16, 9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3-->ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.
The Escherichia coli Fis protein binds to three sites in the upstream activation region of the rrnB P1 promoter and enhances transcription 5- to 10-fold in vivo. In this report, we investigate the mechanism of Fis-dependent activation of transcription. We show that stimulation of rrnB P1 transcription by Fis can occur on linear DNA templates and does not require DNA upstream of the promoter-proximal Fis site I. Mutants of Fis defective for Hin-mediated recombination have been isolated previously and have defined an N-terminal domain required for DNA inversion by Hin in addition to the C-terminal domain which is required for DNA binding. Several of these mutants were found to be defective in stimulation of rrnB P1 transcription in vivo and in vitro. Activation-defective mutants fall into three classes: those that fail to bind to the upstream activation region, those that bind but fail to bend the DNA normally, and those that bind and bend but still fail to activate transcription. We conclude that it is unlikely that Fis functions by simply bringing upstream sequences or bound factors into the proximity of RNA polymerase to activate transcription. Rather, the data are most easily interpreted in terms of transcription activation by direct interactions between Fis and RNA polymerase, requiring precise positioning of the two proteins facilitated by bending of the DNA binding site.
The P1 promoters of the seven Escherichia coli rRNA operons contain recognition sequences for the RNA polymerase (RNAP) holoenzyme containing sigma 70 (E sigma 70), which has been shown to interact with and initiate transcription from rrn P1 promoters in vivo and in vitro. The rrn P1 promoters also contain putative recognition elements for E sigma 32, the RNAP holoenzyme responsible for the transcription of heat shock genes. Using in vitro transcription assays with purified RNAP holoenzyme, we show that E sigma 32 is able to transcribe from the rrnB P1 promoter. Antibodies specific to sigma 70 eliminate transcription of rrnB P1 by E sigma 70 but have no effect on E sigma 32-directed transcription. Physical characterization of the E sigma 32-rrnB P1 complex shows that there are differences in the interactions made by E sigma 70 and E sigma 32 with the promoter. E sigma 32 responds to both Fis-mediated and factor-independent upstream activation, two systems shown previously to stimulate rrnB P1 transcription by E sigma 70. We find that E sigma 32 is not required for two major control systems known to regulate rRNA transcription initiation at normal temperatures in vivo, stringent control and growth rate-dependent control. On the basis of the well-characterized role of E sigma 32 in transcription from heat shock promoters in vivo, we suggest that E sigma 32-directed transcription of rRNA promoters might play a role in ribosome synthesis at high temperatures.
Transcription from the Escherichia coli rrnB P1 promoter is increased by a cis-acting sequence which extends upstream of the -35 hexamer to about -150 with respect to the transcription initiation site, the Upstream Activation Region (UAR). Activation by the UAR involves two components: (1) a trans-acting protein, Fis, which binds to three sites in the UAR between -60 and -150, and (2) the UAR sequences themselves which affect RNA polymerase (RNAP) activity independent of other proteins. We refer to the latter as Factor-Independent Activation (FIA). In addition to its interactions with the -10 and -35 hexamers typical of E. coli promoters, RNAP makes contacts to the -53 region of rrnB P1, which may be related to the FIA effect. We constructed a series of insertion mutants containing integral and non-integral numbers of helical turns at position -46, between the Fis binding sites and the -35 region, and the resulting promoter activities were measured in vitro and in vivo. The data suggest that both Fis-dependent and factor-independent activation are face of the helix dependent: the Fis binding site and the sequences responsible for factor-independent activation must be correctly oriented relative to RNA polymerase in order to activate transcription. These results, in conjunction with other evidence, support a model for the involvement of direct Fis-RNAP interactions in upstream activation. We also demonstrate that RNAP interacts with the -53 region of the rrnB P1 UAR even when these sequences are displaced upstream of the RNAP binding site, and that these interactions correlate with factor-independent activation.
We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.
The conditional lethal mutations ts8 and h8 are located in fda, the gene encoding aldolase, and they inhibit RNA synthesis upon shift to the nonpermissive temperature. We demonstrate that both mutations preferentially inhibit stable RNA synthesis and that this inhibition occurs at the level of transcription initiation. The susceptibility of a promoter to the inhibitory effects of ts8 is correlated with the ability of the promoter to be growth rate regulated. This effect is independent of relA and spoT function. Inhibition is dependent upon glucose metabolism past the generation of glucose-6-phosphate; however, the mechanism of this effect is unknown.
The region from position -154 to position -50 upstream from the start site of transcription of the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), is required for maximal promoter activity in vivo. Maximal activation (20 to 30-fold) requires the binding of Fis protein in vitro and in vivo. However, two- to fourfold activation remains in vivo even in the absence of Fis. Here, we demonstrate that the presence of the UAR increases the rate of formation of E sigma 70-promoter complexes in vitro in the absence of added protein factors (factor-independent activation). The UAR increases the rate of the RNA polymerase concentration-dependent step in the association pathway to a stable complex formed in the presence of the initiating nucleotides ATP and CTP (RPinit). The rate of dissociation from RPinit is not affected. In addition, a supercoiled template of native superhelical density increases both the association rate for the formation of RPinit and the lifetime of complexes formed in the absence of nucleotides (RPo or open complex), but does not affect factor-independent activation. The data are consistent with a model whereby the UAR affects only the initial recognition event (closed complex formation) without affecting either the rate or extent of isomerization to the locally denatured open complex. In the accompanying paper, a variety of chemical and enzymatic probes are used to characterize RPinit and RPo both with and without the UAR.
A region upstream from the Escherichia coli rrnB P1 promoter, the upstream activator region (UAR), increases the activity of the promoter in vivo and the rate of association with RNA polymerase (E sigma 70) in vitro in the presence of the two initiating nucleotides. We have used four types of chemical and enzymatic footprinting probes to determine whether rrnB P1-E sigma 70 complexes formed in the presence of the initiating nucleotides (RPinit) differ from typical open complexes (RPo) formed in the absence of the initiating nucleotides and to examine the structural differences between rrnB P1 complexes containing the UAR and those lacking the UAR. We find that the rrnB P1-RPinit complex closely resembles open complexes formed at other E sigma 70 promoters, indicating that the formation of the first phosphodiester bond does not result in a major rearrangement of the promoter-RNA polymerase complex. An unusual potassium permanganate modification at position -18 in both RPo and RPinit indicates the possible presence of a subtle difference in the -10, -35 spacer structure compared to some other E. coli promoters. We show that the E sigma 70-rrnB P1 complex formed with the promoter containing the UAR has DNase I and hydroxyl radical cleavage patterns in the -50 region different from those observed with the same promoter lacking the UAR. These results are interpreted to indicate that E sigma 70 may interact with a region further upstream from that contacted by RNA polymerase bound at most other promoters and/or that unusual structural properties of this region are induced by bound E sigma 70.
An upstream activation region (UAR) contributes to the extremely high activity of the Escherichia coli ribosomal RNA promoter, rrnB P1, increasing its activity 20- to 30-fold over that of the same promoter lacking the UAR. We have used DNase footprinting to define three specific sites in the rrnB P1 UAR that bind Fis, a protein identified previously by its role in recombinational enhancer function in other systems. We find that purified Fis activates transcription from promoters containing these sites 10- to 20-fold in vitro at concentrations correlating with the filling of these sites. Three approaches indicate that Fis contributes to the function of the UAR in vivo. First, there is a progressive loss in the activity of rrnB P1-lacZ fusions as Fis binding sites are deleted. Second, an rrnB P1 promoter with a mutation in a Fis binding site has 5-fold reduced transcription activity in vivo, dramatically reduced Fis binding in vitro, and shows no Fis dependent transcription activation in vitro. Third, upstream activation is reduced 5-fold in a Fis- strain. We show that rRNA promoters derepress in response to the loss of Fis in vivo in accord with the predictions of the negative feedback model for rRNA regulation. We find that fis is not essential for the function of two control systems known to regulate rRNA, growth rate dependent control and stringent control. On the basis of these results, we propose roles for Fis and the upstream activation system in rRNA synthesis.
Transcription from Escherichia coli ribosomal RNA promoters is increased about 20-fold in vivo by a DNA sequence (the Upstream Activation Region, UAR) located upstream of the -35 conserved hexamer. The UAR stimulates transcription through two mechanisms: one which involves binding of the Fis protein to the UAR, and another mechanisms which functions in the absence of additional protein factors. We have previously constructed a collection of mutations in the region upstream of the -35 hexamer of rrnB P1. Most of these mutations have either no effect on promoter activity or decrease activity 2-5-fold in vivo (Gaal, T., Barkei, J., Dickson, R.R., De Boer, H.A., De Haseth, P.L., Alavi, H. and Gourse, R.L.(1989) J. Bacteriol. 171, 4852-4861). Two mutations leave both the -35 consensus hexamer and the Fis binding consensus sequence intact, yet have larger (14-50-fold) effects on transcription. One substitution just upstream of the -35 hexamer (a C to T change at position -37) primarily affects intrinsic promoter strength, leaving the UAR functional. On the other hand, a three base pair deletion (bases -38 through -40) severely reduces UAR-mediated activity. A substitution covering the three base pair deletion was constructed and found to be activated normally. UAR function appears dependent on its position relative to the RNA polymerase binding site, suggesting that a particular spatial geometry may be necessary for Fis-dependent and/or factor-independent activation to occur.
rRNA synthesis in Escherichia coli is subject to at least two regulation systems, growth rate-dependent control and stringent control. The inverse correlation between rRNA synthesis rates and guanosine 3'-diphosphate 5'-diphosphate (ppGpp) levels under various physiological conditions has led to the supposition that ppGpp is the mediator of both control mechanisms by inhibiting transcription from rrn P1 promoters. Recently, relA- spoT- strains have been constructed in which both ppGpp synthesis pathways most likely have been removed (M. Cashel, personal communication). We have confirmed that such strains produce no detectable ppGpp and therefore offer a direct means for testing the involvement of ppGpp in the regulation of rRNA synthesis in vivo. Stringent control was determined by measurement of rRNA synthesis after amino acid starvation, while growth rate control was determined by measurement of rRNA synthesis under different nutritional conditions. As expected, the relA- spoT- strain is relaxed for stringent control. However, growth rate-dependent regulation is unimpaired. These results indicate that growth rate regulation can occur in the absence of ppGpp and imply that ppGpp is not the mediator, or at least is not the sole mediator, of growth rate-dependent control. Therefore, growth rate-dependent control and stringent control may utilize different mechanisms for regulating stable RNA synthesis.
We measured the activities of 50 operon fusions from a collection of mutant and wild-type rrnB P1 (rrnB1p in the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoters under different nutritional conditions in order to analyze the DNA sequence determinants of growth rate-dependent regulation of rRNA transcription in Escherichia coli. Mutants which deviated from the wild-type -10 or -35 hexamers or from the wild-type 16-base-pair spacer length between the hexamers were unregulated, regardless of whether the mutations brought the promoters closer to the E. coli promoter consensus sequence and increased activity or whether the changes took the promoters further away from the consensus and reduced activity. These data suggest that rRNA promoters have evolved to maintain their regulatory abilities rather than to maximize promoter strength. Some double substitutions outside the consensus hexamers were almost completely unregulated, while single substitutions at several positions outside the -10 and -35 consensus hexamers exerted smaller but significant effects on regulation. These studies suggest roles for specific promoter sequences and/or structures in interactions with regulatory molecules and suggest experimental tests for models of rRNA regulation.
Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p according to the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoter fragments containing single base substitutions, insertions, deletions, and multiple mutations, covering the whole length of the promoter including the upstream activation sequence (UAS). The activities of 112 mutant promoters were assayed as operon fusions to lacZ in lambda lysogens. The activities of most mutants with changes in the core promoter recognition region (i.e., substitutions, insertions, or deletions in the region of the promoter spanning the -10 and -35 E. coli consensus hexamers) correlated with changes toward or away from the consensus in the hexamer sequences or in the spacing between them. However, changes at some positions in the core promoter region not normally associated with transcriptional activity in other systems also had significant effects on rrnB P1. Since rRNA promoter activity varies with cellular growth rate, changes in activity can be the result of changes in promoter strength or of alterations in the regulation of the promoter. The accompanying paper (R. R. Dickson, T. Gaal, H. A. deBoer, P. L. deHaseth, and R. L. Gourse, J. Bacteriol. 171:4862-4870, 1989) distinguishes between these two alternatives. Several mutations in the UAS resulted in two- to fivefold reductions in activity. However, two mutants with changes just upstream of the -35 hexamer in constructs containing the UAS had activities 20- to 100-fold lower than the wild-type level. This collection of mutant rRNA promoters should serve as an important resource in the characterization of the mechanisms responsible for upstream activation and growth rate-dependent regulation of rRNA transcription.
We have established conditions that stabilize the interaction between RNA polymerase and the rrnB P1 promoter in vitro. The requirements for quantitative complex formation are unusual for E. coli promoters: (1) The inclusion of a competitor is required to allow visualization of a specific footprint. (2) Low salt concentrations are necessary since complex formation is salt sensitive. (3) The addition of the initiating nucleotides ATP and CTP, resulting in a low rate of dinucleotide production, is required in order to prevent dissociation of the complexes. The complex has been examined using DNAase I footprinting and filter binding assays. It is characterized by a region protected from DNAase I cleavage that extends slightly upstream of the region protected by RNA polymerase in most E. coli promoters. We find that only one mole of active RNA polymerase is required per mole of promoter DNA in order to detect filter-bound complexes. Under the conditions measured, the rate of association of RNA polymerase with rrnB P1 is as rapid as, or more rapid than, that reported for any other E. coli or bacteriophage promoter.
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