Karen M. Wassarman

Regulation by Small RNAs in Bacteria: Expanding Frontiers

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.

Global analysis of small RNA and mRNA targets of Hfq

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.

The Sm-like Hfq Protein Increases OxyS RNA Interaction with Target mRNAs

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.

Small RNAs in Bacteria: Diverse Regulators of Gene Expression in Response to Environmental Changes

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.

A highly conserved 6S RNA structure is required for regulation of transcription.

6S RNA, a highly abundant noncoding RNA, regulates transcription through interaction with RNA polymerase in Escherichia coli. Computer searches identified 6S RNAs widely among γ-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 σ70-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.

Synthesis-Mediated Release of a Small RNA Inhibitor of RNA Polymerase

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: a regulator of transcription

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.

6S RNA Function Enhances Long-Term Cell Survival

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.

Promoter specificity for 6S RNA regulation of transcription is determined by core promoter sequences and competition for region 4.2 of σ70

6S RNA binds σ70‐RNA polymerase and downregulates transcription at many σ70‐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 σ70 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 σ70.

6S RNA regulation of pspF transcription leads to altered cell survival at high pH.

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.