All organisms contain small RNAs that participate in diverse cellular activities including RNA processing, mRNA stability, translation, protein stability and secretion. These RNAs are defined by their size (< 350 nucleotides in length) and by the fact that they function as an RNA moiety that is not translated into protein. The research in my laboratory focuses on small RNAs in bacteria using molecular, biochemical and genetic approaches to investigate the function and mechanism of action of these RNAs. For example, the 6S RNA is a highly stable and abundant small RNA that associates with and regulates RNA polymerase. Cells with altered levels of 6S RNA show decreased ability to survive stationary phase. Further investigation of 6S RNA action and the genes it regulates has lead to a better understanding of a novel mechanism to control gene expression. In addition, understanding 6S RNA function may provide insights into how cells are able to survive extended nutrient starvation.
There are many new small RNAs in E. coli discovered by genomic and molecular approaches. My laboratory continues to study a number of novel small RNAs to elucidate their roles in the cell. Binding of a common protein (Hfq) has provided hints that some of these small RNAs may regulate translation of target mRNAs under specialized environmental growth conditions. The details of when and how these RNAs act remains to be worked out. In addition, a number of tantalizingly unique small RNAs are present. I anticipate the discovery of numerous additional small RNAs and the revelation of many of their functions will lead to a more complete understanding of the roles small RNAs play in all cells.
Microbiology 726: Regulation of Gene Expression in Prokaryotes
Microbiology 470: Microbial Genetics & Molecular Machines
Trainer, Cellular and Molecular Biology Graduate Program
Trainer, Genetics Graduate Program
Trainer, Microbiology Doctoral Training Program
UW-Madison RNA Maxigroup
Noncoding RNAs (ncRNAs) regulate gene expression in all organisms. Bacterial 6S RNAs globally regulate transcription by binding RNA polymerase (RNAP) holoenzyme and competing with promoter DNA. Escherichia coli (Eco) 6S RNA interacts specifically with the housekeeping σ-holoenzyme (Eσ) and plays a key role in the transcriptional reprogramming upon shifts between exponential and stationary phase. Inhibition is relieved upon 6S RNA-templated RNA synthesis. We report here the 3.8 Å resolution structure of a complex between 6S RNA and Eσdetermined by single-particle cryo-electron microscopy and validation of the structure using footprinting and crosslinking approaches. Duplex RNA segments have A-form C3' endo sugar puckers but widened major groove widths, giving the RNA an overall architecture that mimics B-form promoter DNA. Our results help explain the specificity of Eco 6S RNA for Eσand show how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP.
Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5' untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion.
The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization-defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I-E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression.
6S RNA is a small, noncoding RNA that interacts with the primary holoenzyme form of RNA polymerase. Escherichia coli 6S RNA is a global regulator that downregulates transcription and is important for modulating stress and optimizing survival during nutrient limitation. Studies in diverse organisms suggest a higher complexity in function than previously appreciated. Some bacteria have multiple 6S RNAs that appear to have independent functions. 6S RNA accumulation profiles also are quite divergent and suggest they integrate into cellular networks in a species-specific manner. Nevertheless, in all tested systems the common theme is a role for 6S RNA in survival. Finally, there has been much excitement about the ability of 6S RNA to be used as a template to synthesize product RNAs (pRNAs). This review highlights the details of 6S RNA in E. coli and compares and contrasts 6S RNAs in multiple species.
Small RNAs are integral regulators of bacterial gene expression, the majority of which act posttranscriptionally by basepairing with target mRNAs, altering translation or mRNA stability. 6S RNA, however, is a small RNA that is a transcriptional regulator, acting by binding directly to σ(70)-RNA polymerase (σ(70)-RNAP) and preventing its binding to gene promoters. At the transition from exponential to stationary phase, 6S RNA accumulates and globally downregulates the transcription of hundreds of genes. At the transition from stationary to exponential phase (outgrowth), 6S RNA is released from σ(70)-RNAP, resulting in a fast increase in free σ(70)-RNAP and transcription of many genes. The transition from stationary to exponential phase is sharp, and is thus accessible for experimental study. However, the transition from exponential to stationary phase is gradual and complicated by changes in other factors, making it more difficult to isolate 6S RNA effects experimentally at this transition. Here, we use mathematical modeling and simulation to study the dynamics of 6S RNA-dependent regulation, focusing on transitions in growth mediated by altered nutrient availability. We first show that our model reproduces the sharp increase in σ(70)-RNAP at outgrowth, as well as the behavior of two experimentally tested mutants, thus justifying its use for characterizing the less accessible dynamics of the transition from exponential to stationary phase. We characterize the dynamics of the two transitions for Escherichia coli wild-type, as well as for mutants with various 6S RNA-RNAP affinities, demonstrating that the 6S RNA regulation mechanism is generally robust to a wide range of such mutations, although the level of regulation at single promoters and their resulting expression fold change will be altered with changes in affinity. Our results provide insight into the potential advantage of transcription regulation by 6S RNA, as it enables storage and efficient release of σ(70)-RNAP during transitions in nutrient availability, which is likely to give a competitive advantage to cells encountering diverse environmental conditions.
The 6S RNA is a non-coding small RNA that binds within the active site of housekeeping forms of RNA polymerases (e.g. Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis) and regulates transcription. Efficient release of RNA polymerase from 6S RNA regulation during outgrowth from stationary phase is dependent on use of 6S RNA as a template to generate a product RNA (pRNA). Interestingly, B. subtilis has two 6S RNAs, 6S-1 and 6S-2, but only 6S-1 RNA appears to be used efficiently as a template for pRNA synthesis during outgrowth. Here, we demonstrate that the identity of the initiating nucleotide is particularly important for the B. subtilis RNA polymerase to use RNA templates. Specifically, initiation with guanosine triphosphate (GTP) is required for efficient pRNA synthesis, providing mechanistic insight into why 6S-2 RNA does not support robust pRNA synthesis as it initiates with adenosine triphosphate (ATP). Intriguingly, E. coli RNA polymerase does not have a strong preference for initiating nucleotide identity. These observations highlight an important difference in biochemical properties of B. subtilis and E. coli RNA polymerases, specifically in their ability to use RNA templates efficiently, which also may reflect the differences in GTP and ATP metabolism in these two organisms.
We have discovered that 6S-1 RNA (encoded by bsrA) is important for appropriate timing of sporulation in Bacillus subtilis in that cells lacking 6S-1 RNA sporulate earlier than wild-type cells. The time to generate a mature spore once the decision to sporulate has been made is unaffected by 6S-1 RNA, and, therefore, we propose that it is the timing of onset of sporulation that is altered. Interestingly, the presence of cells lacking 6S-1 RNA in coculture leads to all cell types exhibiting an early-sporulation phenotype. We propose that cells lacking 6S-1 RNA modify their environment in a manner that promotes early sporulation. In support of this model, resuspension of wild-type cells in conditioned medium from ΔbsrA cultures also resulted in early sporulation. Use of Escherichia coli growth as a reporter of the nutritional status of conditioned media suggested that B. subtilis cells lacking 6S-1 RNA reduce the nutrient content of their environment earlier than wild-type cells. Several pathways known to impact the timing of sporulation, such as the skf- and sdp-dependent cannibalism pathways, were eliminated as potential targets of 6S-1 RNA-mediated changes, suggesting that 6S-1 RNA activity defines a novel mechanism for altering the timing of onset of sporulation. In addition, 6S-2 RNA does not influence the timing of sporulation, providing further evidence of the independent influences of these two related RNAs on cell physiology.
6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.
Research on the discovery and characterization of small, regulatory RNAs in bacteria has exploded in recent years. These sRNAs act by base pairing with target mRNAs with which they share limited or extended complementarity, or by modulating protein activity, in some cases by mimicking other nucleic acids. Mechanistic insights into how sRNAs bind mRNAs and proteins, how they compete with each other, and how they interface with ribonucleases are active areas of discovery. Current work also has begun to illuminate how sRNAs modulate expression of distinct regulons and key transcription factors, thus integrating sRNA activity into extensive regulatory networks. In addition, the application of RNA deep sequencing has led to reports of hundreds of additional sRNA candidates in a wide swath of bacterial species. Most importantly, recent studies have served to clarify the abundance of remaining questions about how, when, and why sRNA-mediated regulation is of such importance to bacterial lifestyles.
6S RNA is a small, non-coding RNA that interacts directly with σ(70)-RNA polymerase and regulates transcription at many σ(70)-dependent promoters. Here, we demonstrate that 6S RNA regulates transcription of relA, which encodes a ppGpp synthase. The 6S RNA-dependent regulation of relA expression results in increased ppGpp levels during early stationary phase in cells lacking 6S RNA. These changes in ppGpp levels, although modest, are sufficient to result in altered regulation of transcription from σ(70)-dependent promoters sensitive to ppGpp, including those promoting expression of genes involved in amino acid biosynthesis and rRNA. These data place 6S RNA as another player in maintaining appropriate gene expression as cells transition into stationary phase. Independent of this ppGpp-mediated 6S RNA-dependent regulation, we also demonstrate that in later stationary phase, 6S RNA continues to downregulate transcription in general, and specifically at a subset of the amino acid promoters, but through a mechanism that is independent of ppGpp and which we hypothesize is through direct regulation. In addition, 6S RNA-dependent regulation of σ(S) activity is not mediated through observed changes in ppGpp levels. We suggest a role for 6S RNA in modulating transcription of several global regulators directly, including relA, to downregulate expression of key pathways in response to changing environmental conditions.
No abstract available.
6S RNA is a small, non-coding RNA that interacts with sigma(70)-RNA polymerase and downregulates transcription at many promoters during stationary phase. When bound to sigma(70)-RNA polymerase, 6S RNA is engaged in the active site of sigma(70)-RNA polymerase in a manner similar enough to promoter DNA that the RNA can serve as a template for RNA synthesis. It has been proposed that 6S RNA mimics the conformation of DNA during transcription initiation, suggesting contacts between RNA polymerase and 6S RNA or DNA may be similar. Here we demonstrate that region 4.2 of sigma(70) is critical for the interaction between 6S RNA and RNA polymerase. We define an expanded binding surface that encompasses positively charged residues throughout the recognition helix of the helix-turn-helix motif in region 4.2, in contrast to DNA binding that is largely focused on the N-terminal region of this helix. Furthermore, negatively charged residues in region 4.2 weaken binding to 6S RNA but do not similarly affect DNA binding. We propose that the binding sites for promoter DNA and 6S RNA on region 4.2 of sigma(70) are overlapping but distinct, raising interesting possibilities for how core promoter elements contribute to defining promoters that are sensitive to 6S RNA regulation.
6S RNA binds sigma70-RNA polymerase and downregulates transcription at many sigma70-dependent promoters, but others escape regulation even during stationary phase when the majority of the transcription machinery is bound by the RNA. We report that core promoter elements determine this promoter specificity; a weak -35 element allows a promoter to be 6S RNA sensitive, and an extended -10 element similarly determines 6S RNA inhibition except when a consensus -35 element is present. These two features together predicted that hundreds of mapped Escherichia coli promoters might be subject to 6S RNA dampening in stationary phase. Microarray analysis confirmed 6S RNA-dependent downregulation of expression from 68% of the predicted genes, which corresponds to 49% of the expressed genes containing mapped E. coli promoters and establishes 6S RNA as a global regulator in stationary phase. We also demonstrate a critical role for region 4.2 of sigma70 in RNA polymerase interactions with 6S RNA. Region 4.2 binds the -35 element during transcription initiation; therefore we propose one mechanism for 6S RNA regulation of transcription is through competition for binding region 4.2 of sigma70.
The past decade has seen an explosion in discovery of small, non-coding RNAs in all organisms. As functions for many of the small RNAs have been identified, it has become increasingly clear that they are important components in regulating gene expression. A multitude of RNAs target mRNAs for regulation at the level of translation or stability, including the microRNAs in higher eukaryotes and the Hfq binding RNAs in bacteria. Other RNAs regulate transcription, such as murine B2 RNA, mammalian 7SK RNA and the bacterial 6S RNA, which will be the focus of this review. Details of 6S RNA interactions with RNA polymerase, how 6S RNA regulates transcription, and how 6S RNA function contributes to cellular survival are discussed.
Appreciation for the prevalence and diversity of noncoding, small RNAs (sRNAs) has grown enormously in the past decade. A major role for sRNAs in all organisms is to regulate gene expression, often at the level of mRNA translation or stability. However, a few sRNAs have been shown to function by regulating transcription. The bacterial 6S RNA was the first sRNA shown to inhibit transcription by binding directly to the housekeeping holoenzyme form of RNA polymerase (i.e. sigma70-RNA polymerase in E. coli). It resides within the active site of RNA polymerase, blocks access to promoter DNA and, surprisingly, is used as a template for RNA synthesis. 6S RNA regulation of transcription leads to altered cell survival, perhaps by redirecting resource utilization under nutrient-limiting conditions.
In recent years, the combinations of computational and molecular approaches have led to the identification of an increasing number of small, noncoding RNAs encoded by bacteria and their plasmids and phages. Most of the characterized small RNAs have been shown to operate at a posttranscriptional level, modulating mRNA stability or translation by base-pairing with the 5' regions of the target mRNAs. However, a subset of small RNAs has been found to regulate transcription. One example is the abundant 6S RNA that has been proposed to compete for DNA binding of RNA polymerase by mimicking the open conformation of promoter DNA. Other small RNAs affect transcription termination via base-pairing interactions with sequences in the mRNA. Here, we discuss current understanding and questions regarding the roles of small RNAs in regulating transcription.
Noncoding small RNAs regulate gene expression in all organisms, in some cases through direct association with RNA polymerase (RNAP). Here we report that the mechanism of 6S RNA inhibition of transcription is through specific, stable interactions with the active site of Escherichia coli RNAP that exclude promoter DNA binding. In fact, the DNA-dependent RNAP uses bound 6S RNA as a template for RNA synthesis, producing 14-to 20-nucleotide RNA products (pRNA). These results demonstrate that 6S RNA is functionally engaged in the active site of RNAP. Synthesis of pRNA destabilizes 6S RNA-RNAP complexes leading to release of the pRNA-6S RNA hybrid. In vivo, 6S RNA-directed RNA synthesis occurs during outgrowth from the stationary phase and likely is responsible for liberating RNAP from 6S RNA in response to nutrient availability.
6S RNA is a highly abundant small RNA that regulates transcription through direct interaction with RNA polymerase. Here we show that 6S RNA directly inhibits transcription of pspF, which subsequently leads to inhibition of pspABCDE and pspG expression. Cells without 6S RNA are able to survive at elevated pH better than wild-type cells due to loss of 6S RNA-regulation of pspF. This 6S RNA-dependent phenotype is eliminated in pspF-null cells, indicating that 6S RNA effects are conferred through PspF. Similar growth phenotypes are seen when PspF levels are increased in a 6S RNA-independent manner, signifying that changes to pspF expression are sufficient. Changes in survival at elevated pH most likely result from altered expression of pspABCDE and/or pspG, both of which require PspF for transcription and are indirectly regulated by 6S RNA. 6S RNA provides another layer of regulation in response to high pH during stationary phase. We propose that the normal role of 6S RNA at elevated pH is to limit the extent of the psp response under conditions of nutrient deprivation, perhaps facilitating appropriate allocation of diminishing resources.
6S RNA, a highly abundant noncoding RNA, regulates transcription through interaction with RNA polymerase in Escherichia coli. Computer searches identified 6S RNAs widely among gamma-proteobacteria. Biochemical approaches were required to identify more divergent 6S RNAs. Two Bacillus subtilis RNAs were found to interact with the housekeeping form of RNA polymerase, thereby establishing them as 6S RNAs. A third B. subtilis RNA was discovered with distinct RNA polymerase-binding activity. Phylogenetic comparison and analysis of mutant RNAs revealed that a conserved secondary structure containing a single-stranded central bulge within a highly double-stranded molecule was essential for 6S RNA function in vivo and in vitro. Reconstitution experiments established the marked specificity of 6S RNA interactions for sigma(70)-RNA polymerase, as well as the ability of 6S RNA to directly inhibit transcription. These data highlight the critical importance of structural characteristics for 6S RNA activity.
6S RNA was identified in Escherichia coli >30 years ago, but the physiological role of this RNA has remained elusive. Here, we demonstrate that 6S RNA-deficient cells are at a disadvantage for survival in stationary phase, a time when 6S RNA regulates transcription. Growth defects were most apparent as a decrease in the competitive fitness of cells lacking 6S RNA. To decipher the molecular mechanisms underlying the growth defects, we have expanded studies of 6S RNA effects on transcription. 6S RNA inhibition of sigma(70)-dependent transcription was not ubiquitous, in spite of the fact that the vast majority of sigma(70)-RNA polymerase is bound by 6S RNA during stationary phase. The sigma(70)-dependent promoters inhibited by 6S RNA contain an extended -10 promoter element, suggesting that this feature may define a class of 6S RNA-regulated genes. We also discovered a secondary effect of 6S RNA in the activation of sigma(S)-dependent transcription at several promoters. We conclude that 6S RNA regulation of both sigma(70) and sigma(S) activities contributes to increased cell persistence during nutrient deprivation.
Hfq, a bacterial member of the Sm family of RNA-binding proteins, is required for the action of many small regulatory RNAs that act by basepairing with target mRNAs. Hfq binds this family of small RNAs efficiently. We have used co-immunoprecipitation with Hfq and direct detection of the bound RNAs on genomic microarrays to identify members of this small RNA family. This approach was extremely sensitive; even Hfq-binding small RNAs expressed at low levels were readily detected. At least 15 of 46 known small RNAs in E. coli interact with Hfq. In addition, high signals in other intergenic regions suggested up to 20 previously unidentified small RNAs bind Hfq; five were confirmed by Northern analysis. Strong signals within genes and operons also were detected, some of which correspond to known Hfq targets. Within the argX-hisR-leuT-proM operon, Hfq appears to compete with RNase E and modulate RNA processing and degradation. Thus Hfq immunoprecipitation followed by microarray analysis is a highly effective method for detecting a major class of small RNAs as well as identifying new Hfq functions.
No abstract available.
Bacterial small, untranslated RNAs are important regulators that often act to transmit environmental signals when cells encounter suboptimal or stressful growth conditions. These RNAs help modulate changes in cellular metabolism to optimize utilization of available nutrients and improve the probability for survival.
The Escherichia coli host factor I, Hfq, binds to many small regulatory RNAs and is required for OxyS RNA repression of fhlA and rpoS mRNA translation. Here we report that Hfq is a bacterial homolog of the Sm and Sm-like proteins integral to RNA processing and mRNA degradation complexes in eukaryotic cells. Hfq exhibits the hallmark features of Sm and Sm-like proteins: the Sm1 sequence motif, a multisubunit ring structure (in this case a homomeric hexamer), and preferential binding to polyU. We also show that Hfq increases the OxyS RNA interaction with its target messages and propose that the enhancement of RNA-RNA pairing may be a general function of Hfq, Sm, and Sm-like proteins.
A burgeoning list of small RNAs with a variety of regulatory functions has been identified in both prokaryotic and eukaryotic cells. However, it remains difficult to identify small RNAs by sequence inspection. We used the high conservation of small RNAs among closely related bacterial species, as well as analysis of transcripts detected by high-density oligonucleotide probe arrays, to predict the presence of novel small RNA genes in the intergenic regions of the Escherichia coli genome. The existence of 23 distinct new RNA species was confirmed by Northern analysis. Of these, six are predicted to encode short ORFs, whereas 17 are likely to be novel functional small RNAs. We discovered that many of these small RNAs interact with the RNA-binding protein Hfq, pointing to a global role of the Hfq protein in facilitating small RNA function. The approaches used here should allow identification of small RNAs in other organisms.
The E. coli 6S RNA was discovered more than three decades ago, yet its function has remained elusive. Here, we demonstrate that 6S RNA associates with RNA polymerase in a highly specific and efficient manner. UV crosslinking experiments revealed that 6S RNA directly contacts the sigma70 and beta/beta' subunits of RNA polymerase. 6S RNA accumulates as cells reach the stationary phase of growth and mediates growth phase-specific changes in RNA polymerase. Stable association between sigma70 and core RNA polymerase in extracts is only observed in the presence of 6S RNA. We show 6S RNA represses expression from a sigma70-dependent promoter during stationary phase. Our results suggest that the interaction of 6S RNA with RNA polymerase modulates sigma70-holoenzyme activity.
Bacterial cells contain several small RNAs (sRNAs) that are not translated. These stable, abundant RNAs act by multiple mechanisms, such as RNA-RNA basepairing, RNA-protein interactions and intrinsic RNA activity, and regulate diverse cellular functions, including RNA processing, mRNA stability, translation, protein stability and secretion.