Summary 6S RNA binds s 70-RNA polymerase and downregulates transcription at many s 70-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 s 70 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 s 70.

6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ70 in Escherichia coli, EσA in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ70 during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ70 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 EσA 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.

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.

6S RNA is a small, non-coding RNA that interacts directly with σ70-RNA polymerase and regulates transcription at many σ70-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 σ70-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 σS 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.

The 6S RNA is a non-coding small RNA that binds within the active site of housekeeping forms of RNA polymerases (e.g. Eσ70 in Escherichia coli, EσA 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.