6S RNA, a Global Regulator of Transcription

ABSTRACT

6S RNA is a small RNA regulator of RNA polymerase (RNAP) that is present broadly throughout the bacterial kingdom. Initial functional studies in Escherichia coli revealed that 6S RNA forms a complex with RNAP resulting in regulation of transcription, and cells lacking 6S RNA have altered survival phenotypes. The last decade has focused on deepening the understanding of several aspects of 6S RNA activity, including (i) addressing questions of how broadly conserved 6S RNAs are in diverse organisms through continued identification and initial characterization of divergent 6S RNAs; (ii) the nature of the 6S RNA-RNAP interaction through examination of variant proteins and mutant RNAs, cross-linking approaches, and ultimately a cryo-electron microscopic structure; (iii) the physiological consequences of 6S RNA function through identification of the 6S RNA regulon and promoter features that determine 6S RNA sensitivity; and (iv) the mechanism and cellular impact of 6S RNA-directed synthesis of product RNAs (i.e., pRNA synthesis). Much has been learned about this unusual RNA, its mechanism of action, and how it is regulated; yet much still remains to be investigated, especially regarding potential differences in behavior of 6S RNAs in diverse bacteria.

INTRODUCTION

It is now well established that small RNAs (sRNAs) have diverse and widespread roles in regulating gene expression in all organisms (1–4). Mechanisms of action are varied but can be broadly classified into three categories: (i) sRNAs that act by base-pairing to target RNAs; (ii) sRNAs that act to modulate protein activity through direct RNA-protein interaction; and (iii) sRNAs that have intrinsic function (e.g., catalytic). Two well-studied sRNA families that modulate protein activity include sRNAs that regulate CsrA protein and 6S RNA, which regulates RNA polymerase (RNAP) and is the focus here as well as in several other reviews (5–8). 6S RNA was first identified in Escherichia coli (9, 10), which remains the best-understood model of 6S RNA function, although identification of 6S RNAs and their roles in diverse bacterial species has been an active area of research in the past decade. Here, information for E. coli will be presented first, followed by discussion of similarities and differences known or postulated for 6S RNAs in diverse species.

E. coli 6S RNA (Ec6S RNA) was first discovered as a highly abundant, stable RNA more than 50 years ago (9), but it was not until the discovery that Ec6S RNA formed a complex with RNAP more than 30 years later that its biological function and mechanism of action began to be revealed (11). Bacterial RNAP is a multisubunit enzyme, consisting of a core (E: ααββ′) that is transcriptionally competent but requires the addition of a specificity factor (sigma: σ) to form the holoenzyme (Eσ) needed to recognize promoters and initiate transcription (12, 13). All bacteria contain a housekeeping σ, usually referred to as σ⁷⁰ or σ^A, which is highly abundant and required at all stages of growth. Different bacterial species have varied numbers of alternative sigma factors, ranging from zero to several dozen. Ec6S RNA interacts specifically and very tightly with the housekeeping holoenzyme form of RNAP (Eσ⁷⁰ in E. coli) (11, 14). Early studies demonstrated that Ec6S RNA binding to Eσ⁷⁰ resulted in downregulation of σ⁷⁰-dependent transcription at a tested promoter (rsdP2) and that cells lacking Ec6S RNA have altered survival phenotypes (11, 15, 16). These findings led to the suggestion that the physiological role of 6S RNA, at least in E. coli, is to contribute to regulation of gene expression in response to poor nutrient environments. Research over the past decade has brought considerable insight into 6S RNA function but also raised many questions for future work. This review focuses on several active areas of inquiry including the identification of 6S RNAs in diverse bacterial species; the details of 6S RNA-RNAP interactions; the identification of promoters regulated by 6S RNA; the physiological consequences of 6S RNA-dependent regulation; and finally the discovery and impact of product RNA (pRNA) synthesis, a process in which 6S RNA serves as a template for synthesis of a pRNA. The biogenesis of Ec6S RNA has also been an area of interest and active research but will not be discussed here. For information about the biogenesis of 6S RNA, see references 5 and 17–24.

6S RNAs ARE WIDESPREAD THROUGHOUT THE BACTERIAL KINGDOM

Identification of 6S RNA and Candidate 6S RNAs

During the past 15 years, much work on 6S RNA has focused on its interaction with Eσ⁷⁰ and its presence and potential impact in diverse bacteria. Biochemical and phylogenetic approaches demonstrated that overall secondary structure was critical for 6S RNA-Eσ⁷⁰ interactions (14, 25). Specifically, 6S RNA is primarily double stranded with minor disruptions of bulged nucleotides in addition to a large, central, single-stranded region (Fig. 1). It is this secondary structure, rather than sequence identity, that is required for Ec6S RNA to bind Eσ⁷⁰ and has been the basis for identification of most 6S RNAs and 6S RNA-encoding genes in diverse genomic sequences. Alternative approaches to identify 6S RNAs have included identification of sRNAs that coimmunoprecipitate with RNAP or through identification of 6S RNA-associated pRNA in RNA-sequencing data. Altogether, 6S RNAs have been identified or predicted in a wide range of bacteria (26), making it one of the very highly conserved sRNAs along with transfer-messenger RNA (tmRNA), signal recognition particle RNA, and the Csr/Rsm family of RNAs (27).

FIGURE 1.

E. coli 6S RNA is shown in a secondary structure observed from the cryo-EM 6S RNA-Eσ⁷⁰ structure (upstream stem and central region) or predicted from secondary-structure analysis (downstream stem) (14, 61). For reference, the upstream stem, central region, and downstream stem are indicated. The template central region is in green; the site of initiation for pRNA synthesis is indicated by a red arrow; the nontemplate central region is in blue. 6S RNAs from B. subtilis (Bs6S-1 and Bs6S-2), H. pylori, L. pneumophila (Lp6S RNA), R. sphaeroides, and A. aeolicus are shown in secondary structures expected when in complex with RNAP, with sites of pRNA synthesis initiation indicated by arrows (14, 25, 30, 48, 57, 70, 71). pRNA synthesis has not been examined for Lp6S RNA. Note, some 6S RNAs have been demonstrated or predicted to have base pairing within the central region in isolated RNA (Bs6S-1 and Bs6S-2 RNA, Hp6S RNA) (25, 57, 73, 79), but binding studies support that the central region is fully single stranded when bound to RNAP (14, 34, 63, 72). Other alternative structures that contribute to 6S RNA release from RNAP during pRNA synthesis also have been observed (73, 79–81).

Identification of additional 6S RNAs is an ongoing process as more genome sequences become available and as transcriptome sequencing (RNA-seq) approaches to examine RNAs globally are applied to more organisms. However, identification of 6S RNAs globally remains nontrivial, as the parameters defining the structural requirements for interaction with RNAP are not as fixed as structural requirements of some other sRNAs (e.g., tmRNA interaction with ribosomes and alanyl-tRNA synthetases [28]), and recent work suggests that there may be more variation in specific contacts in divergent bacteria than previously predicted. Nevertheless, identification and characterization of diverse 6S RNAs continues to provide important information about 6S RNA and its cellular role generally and in specific species.

Some Species Have Multiple 6S RNAs

Some genomes encode more than one 6S RNA, most notably Bacillus subtilis, where the two 6S RNAs (Bs6S-1 and Bs6S-2 RNAs) (14, 25) have been studied in some detail. Legionella pneumophila also has been reported to express two 6S RNAs, Lp6S RNA and Lp6S-2 RNA, and two candidate 6S RNA-encoding genes were identified in Hydrogenivirga (29–32). The presence of two 6S RNAs in divergent bacteria raises interesting questions regarding how common it is to have multiple 6S RNAs, how they are independently regulated, if their activities are redundant or overlapping, and so forth. In B. subtilis and L. pneumophila, the two 6S RNAs act independently of each other; have different accumulation profiles; and, at least for B. subtilis, are known to regulate different genes (14, 25, 33–35; A. T. Cavanagh and K. M. Wassarman, unpublished data).

6S RNA Accumulation Profiles—Insight into Function?

One of the first steps in characterization of many of the newly identified 6S RNAs has been to look at their expression profiles, especially during growth phase or under specialized growth conditions. Ec6S RNA is present at all times of growth in E. coli, but is at lower levels in early exponential phase (<1,000 copies per cell) and gradually accumulates during growth until it reaches maximal levels (∼10,000 copies per cell) several hours after transition into stationary phase (11). Thus, much work on Ec6S RNA has focused on its role in stationary phase, where it contributes to cell survival, although it should be noted that Ec6S RNA is present in exponential phase, is bound to Eσ⁷⁰, and can regulate transcription at this time (14, 36, 37). In contrast, many 6S RNAs in divergent species have different expression profiles, which may provide insight into predictions of different physiological roles for these RNAs.

Bs6S-1 RNA is an example of a 6S RNA that accumulates in stationary phase with expression profiles similar to Ec6S RNA, and Lp6S RNA also accumulates postexponentially (29, 38). In contrast, Bs6S-2 RNA levels change only modestly throughout growth (<2- to 3-fold), with maximal levels observed in late exponential phase, although the precise expression profile reported for Bs6S-2 RNA has varied between different studies, likely due to differences in strains and growth media and timing of “stationary” phase examined (14, 25, 39). The Lp6S-2 RNA expression profile is more complex but is not similar to Ec6S RNA (31). The differences in accumulation of Bs6S-1 and Bs6S-2 RNAs or Lp6S and Lp6S-2 RNAs suggest that they likely have different physiological roles, in agreement with observations that gene expression and proteomic profiles for cells lacking Bs6S-1 and Bs6S-2 RNAs are different (35; Cavanagh and Wassarman, unpublished), and observed mutant phenotypes (i.e., altered timing of sporulation and cell density changes in stationary phase) are associated specifically with the loss of Bs6S-1 RNA (33, 35). Streptomyces coelicolor 6S RNA also accumulates with a profile similar to Ec6S RNA and influences growth rate (40).

Other 6S RNAs accumulate with different profiles that hint at interesting cellular roles. For example, within identified Cyanobacteria 6S RNAs there is an array of different accumulation patterns. Levels of Prochlorococcus MED4 6S RNA are cell cycle dependent and change with light, suggesting that 6S RNA may contribute to the high light adaptation of this strain (41). 6S RNA in Synechocystis sp. strain PCC 6803 has also been suggested to contribute to light stress as well as recovery from nitrogen depletion (6, 42). Alternatively, 6S RNA in Synechococcus sp. strain PCC 6301 changes during growth, suggesting a response to nutrient status, although in this case 6S RNA is abundant in exponential phase and reduced in stationary phase, perhaps more similar to Bs6S-2 RNA (43).

Examples of several alphaproteobacteria that associate with host cells exhibit differential expression dependent on host association. For example, the plant symbiont Bradyrhizobium japonicum 6S RNA is higher in root nodules compared to free-living cells (44, 45). Wolbachia 6S RNA levels change with host identity (i.e., 6S RNA levels were higher in germ line cells compared to somatic cells) (45, 46). It has been suggested that the Wolbachia 6S RNA accumulation increases during fast replication, in contrast to Ec6S and Bs6S-1 RNAs, which accumulate during slow growth (i.e., stationary phase). Rhodobacter sphaeroides and Caulobacter crescentus (free-living alphaproteobacteria) 6S RNA accumulation patterns change with cell growth, although the change between exponential- and stationary-phase levels is rather modest (∼3-fold) (47, 48; Cavanagh and Wassarman, unpublished). R. sphaeroides 6S RNA has been associated with high-salt-stress survival (47).

Several 6S RNAs from pathogenic bacteria or close relatives have been shown to increase under stress, suggesting a potential role in pathogenesis or in host survival. Examples include Burkholderia cenocepacia 6S RNA, which increases during oxidative stress (49); Yersinia pestis 6S RNA, which has altered levels during lung infection (50); Rickettsia 6S RNA, which accumulates many hours postinfection and correlates with intracellular growth kinetics (51); Coxiella burnetii 6S RNA, which accumulates in the stress-resistant cellular form (small cell variant; SCV), suggesting a role in stress (52); Salmonella enterica serovar Typhimurium 6S RNA, which accumulates at low pH, resulting in altered invasion and stress survival (53); Clostridium acetobutylicum 6S RNA, which increases in response to general stress and is reported to promote butanol tolerance (54); and Borrelia burgdorferi 6S RNA, which accumulates in ticks with timing suggesting a role in persistence (S. Samuels, L. Hall, and D. Drecktrah, personal communication).

Additional 6S RNAs have been observed to be expressed under at least one condition (e.g., Bordetella pertussis [14], C. crescentus [55], Clostridium difficile [56], Helicobacter pylori [57], Pseudomonas aeruginosa [58], and Rhodopseudomonas palustris [45]), and many others have been predicted from genomic sequences with minimal additional information about expression patterns or function yet available. In fact, 6S RNA candidates have been predicted in the majority of bacterial genomes; see reference 26 for a recent update on the distribution of 6S RNAs throughout bacteria.

Many questions remain about 6S RNAs from diverse bacteria, and future work is anticipated to focus on providing further understanding of their physiological roles in different biological circumstances. In addition, there is the potential that some of these 6S RNAs and candidate 6S RNAs may expand on known mechanistic activities.

How To Define Divergent 6S RNA Candidates as 6S RNAs?

Of particular importance as these divergent 6S RNAs are further studied and as additional 6S RNAs candidates are identified is the question of whether these candidates are all true 6S RNAs and what defines a 6S RNA. We have previously suggested that 6S RNAs should be defined as RNAs that bind their cognate primary holoenzyme form of RNAP in a manner resembling promoter DNA binding, a definition likely to capture a class of RNAs that are mechanistically similar and thereby providing a functionally useful definition (5). It has been suggested to include “directing pRNA synthesis” in the definition (35), but it is not included here, as pRNA synthesis is not required for 6S RNA to regulate transcription (34) and there may be examples where 6S RNAs rely on other mechanisms to cycle off of RNAP but retain similar mechanisms of regulation of transcription. Nevertheless, either definition requires detailed information about RNAP binding, a characteristic that is harder to test, especially in divergent systems where tools are not readily available. In some tested cases, however, there has been quite a large variation in the fraction of 6S RNA that is bound to RNAP (>75% for Ec6S RNA, Bs6S-1 RNA, and Bs6S-2 RNA, compared to <10% for Lp6S RNA) (11, 14, 29), which may be due to technical reasons but could represent critical differences in 6S RNA function. As more details about 6S RNA-RNAP interactions in diverse bacteria become available, it will be important to revisit the definition of 6S RNAs, in particular whether there are additional classes of sRNAs that interact with RNAP to function using different mechanisms.

At least one RNA identified based on its structural similarity to 6S RNA (Ms1 RNA in Mycobacterium smegmatis) does not bind to the holoenzyme form of RNAP, although it does interact with core RNAP (59, 60). This example serves as a cautionary tale in relying too heavily on secondary structure as the predictor of 6S RNA candidates, but also raises the intriguing possibility that there are additional classes of sRNAs that function through interaction with RNAP using different mechanisms. Future work must focus on characterization of 6S RNA candidates beyond secondary-structure predictions for definitive 6S RNA identification.

THE 6S RNA-RNAP COMPLEX

Ec6S RNA-Eσ⁷⁰ Interactions

The past decade has revealed much about the nature of the 6S RNA-RNAP interaction, including a cryo-electron microscopic (cryo-EM) structure of the E. coli 6S RNA-Eσ⁷⁰ complex in the past year (61). As noted above, it has been appreciated for some time that the secondary structure is the key element required for binding, and that there is minimal if any sequence specificity to the interaction (14, 25, 62). It was demonstrated that the central bubble of Ec6S RNA resides near the active site, similar to promoter DNA during transcription initiation when the DNA surrounding the start site of transcription is melted to form the “open complex” (63). Consistent with this hypothesis, the Ec6S RNA can be used as a template by Eσ⁷⁰ to generate pRNA in a process called pRNA synthesis (63). More about pRNA synthesis below, but the ability to use 6S RNA as a template for pRNA synthesis in vitro strongly supported similar binding of 6S RNA and promoter DNA to Eσ⁷⁰ globally. Site-directed cross-link mapping (61) and Fe-BABE [iron(S)-1-(p-bromoacetamidobenzyl) EDTA] cleavage mapping (64) further supported a strong correlation between the path of RNA and DNA when bound to Eσ⁷⁰. The cryo-EM structure provided direct visualization of the 6S RNA-Eσ⁷⁰ complex and its overall similarity to open complexes (61). However, in spite of the overall similarity of architecture, closer inspection also revealed regions where Ec6S RNA and promoter DNA binding were quite different.

Template strand central region

One area of great interest is the template strand of the central region in 6S RNA and how similarly it is positioned in RNAP to the open complex DNA template bubble (65), as this is the region where RNA synthesis initiates during both pRNA synthesis and transcription. Perhaps unsurprisingly, the paths of RNA and DNA are quite similar here, as demonstrated by cross-linking to the same residues and directly visualized in the cryo-EM Ec6S RNA-Eσ⁷⁰ structure compared to open complex structures, although the most upstream single-stranded region of 6S RNA was not as well resolved in the Ec6S RNA-Eσ⁷⁰ structure as the rest of the RNA (61).

Nontemplate central region

The cryo-EM structure also provided information about the path of the nontemplate central region of the 6S RNA. Although most of the nontemplate single-stranded region was similar between Ec6S RNA and open complex DNA, one noted difference is that A131 in Ec6S RNA, the position equivalent to −11A in promoter DNA, was not flipped and thus did not make interactions with the σ⁷⁰ pocket (66). 6S RNA is a premelted bubble and thus is not expected to require this interaction to initiate and maintain melting, in contrast to open complex formation, where flipping of −11A to interact with σ⁷⁰ is thought to initiate melting and recognition of the −10 element during open complex formation (67). Interestingly, the position equivalent to A131 in Ec6S RNA is highly conserved in identified and predicted 6S RNAs, raising questions about whether there is another role for this nucleotide or if there are conditions when it does interact with σ⁷⁰ region 2. However, A131 is not required for Ec6S RNA activity, as mutation of this residue has no detectable effect on the kinetics of binding to Eσ⁷⁰ nor on the efficiency of pRNA synthesis (61).

Other contacts between the nontemplate bubble region of Ec6S RNA (e.g., U135, G136, and G143) and Eσ⁷⁰ were similar to contacts between DNA and Eσ⁷⁰ in the open complex (61). Intriguingly, however, these contacts likewise are not required for 6S RNA binding to Eσ⁷⁰ nor for efficient pRNA synthesis initiation (61), raising questions about whether they play a role in other aspects of 6S RNA function/activity. Sequences in this region do contribute to timing of pRNA synthesis-mediated release of Ec6S RNA from Eσ⁷⁰ through a releasing structure.

Upstream stem interactions with σ⁷⁰ region 4.2

One area where Ec6S RNA interactions are quite different than promoter DNA open complexes is the nucleic acid interaction with region 4.2 of σ⁷⁰ (Fig. 2). The fact that this interaction was distinct between DNA and RNA was first revealed by differential binding of Eσ⁷⁰ variants with alanine substitutions in region 4.2 of σ⁷⁰, which suggested a larger binding surface for 6S RNA (68). The cryo-EM structure provides a high-resolution view of the Ec6S RNA interaction with Eσ⁷⁰ in this region and correlates well with the biochemical analysis (61). The cryo-EM structure also demonstrates an unusual structure for the Ec6S RNA in this region, an area where secondary-structure mapping and mutagenesis had been largely uninformative (Cavanagh and Wassarman, unpublished), consistent with the fact that these techniques are best at predicting canonical structures. The importance of this region of Ec6S RNA for binding to Eσ⁷⁰ was also highlighted by random mutagenesis studies (62).

FIGURE 2.

Comparison of 6S RNA and promoter DNA interactions with region 4.2 of σ⁷⁰. Holoenzyme structures complexed with RNA (PDB ID: 5VT0) (61) (A and C) and promoter DNA (PDB ID: 5VI5) (83) (B and D) are centered on region 4.2 of σ⁷⁰ and shown without (A and B) or with (C and D) the nucleic acids visible. The cryo-EM structure with 6S RNA is E. coli holoenzyme, and the crystal structure with DNA in an open complex is M. smegmatis holoenzyme, and thus there are small variations in structure due to sequence changes, although region 4.2 of σ⁷⁰ is very highly conserved. Residues within region 4.2 of σ⁷⁰ are labeled (E. coli numbering) with color coding based on impact of alanine substitution on binding to RNA (A) or DNA (B) (68): red, strong decrease; blue, moderate decrease; green, increase. A592 (dark gray) is a position where substitution of a positive or negative residue strongly influences 6S RNA binding with little effect on DNA binding (68). Residues labeled in light gray (B and D) are locations where alanine substitution did not influence DNA binding, but are included to assist with comparison of the two structures. Other coloring: 6S RNA, green; promoter DNA, purple; β subunit, cyan; β′ subunit, pink; σ⁷⁰ (outside of region 4.2), light orange.

Aspects of 6S RNA structure allow it to adopt an overall architecture that follows the B-form DNA helix of the open complex

Of particular interest from an RNA structure perspective is the region of RNA between the central bubble and the contacts with region 4.2 of σ⁷⁰, which follows the path and overall architecture of double-stranded, B-form helix DNA rather closely (61). Double-stranded RNA is typically an A-form helix, but the 6S RNA uses a combination of short A-form helices that have some B-form characteristics, in addition to gaps and bulged nucleotides, to adopt this unusual structural mimic of double-stranded DNA.

6S RNA-RNAP Interactions in Diverse Bacteria

Although understanding of the Ec6S RNA-Eσ⁷⁰ interaction has increased substantially over the past decade and has provided much insight into how 6S RNAs function mechanistically, detailed biochemical analysis of 6S RNA-RNAP interactions in diverse bacteria remains largely unexplored. In many organisms, 6S RNA-RNAP complexes have been detected by coimmunoprecipitation or in vitro analysis (e.g., B. subtilis [14, 34, 69], L. pneumophila [29], C. burnetii [52], Aquifex aeolicus [70], and S. coelicolor [40]), supporting a general similarity to Ec6S RNA. However, detailed understanding of interactions in other organisms is likely to require biochemical or structural analysis to uncover any potential differences from Ec6S RNA. Directed in vitro studies have been done for B. subtilis 6S RNAs, revealing many interesting aspects of these RNAs, much of which is focused on pRNA synthesis initiation and release. Most studies to date have assumed that key features of Ec6S RNA-Eσ⁷⁰ interactions are conserved; however, some observations suggest that this assumption may be premature. Some 6S RNAs, notably from alphaproteobacteria and A. aeolicus, have a shortened upstream stem and therefore are lacking the region critical for Ec6S RNA interactions with σ⁷⁰ region 4.2 (25, 68, 71). However, pRNAs have been detected for A. aeolicus and R. sphaeroides 6S RNAs, strongly supporting an RNAP interaction, and the Aa6S RNA has been demonstrated to bind B. subtilis Eσ^A in vitro (47, 70). The H. pylori 6S RNA appears to bind RNAP in either the “forward” or “reverse” orientation based on evidence of pRNAs templated from both strands of the central region (57), in contrast to Ec6S RNA, which binds quite specifically in one orientation and directs pRNA from one strand of the central region (63). Orientation of Ec6S RNA binding to Eσ⁷⁰ has been proposed to be directed through upstream stem contacts with σ⁷⁰ region 4.2; thus, lack of orientation also suggests a change in the requirement or nature of interactions with σ⁷⁰ region 4.2. Additionally, 6S RNA-dependent changes in transcription appear to be different in several tested organisms compared to E. coli, perhaps suggesting key or substantial differences in binding that impact regulation of transcription. Likewise, until the potential differences and similarities between divergent 6S RNAs are understood, caution is advised for studies using noncognate 6S RNA-RNAP matches for in vitro studies, as species-specific behaviors of RNAP in 6S RNA function have been observed (72).

6S RNA REGULATION OF TRANSCRIPTION

Regulation of Transcription in E. coli

The binding of 6S RNA to Eσ⁷⁰, the major transcriptional machinery of the cell, strongly suggested that 6S RNA would regulate σ⁷⁰-dependent transcription, as initially confirmed at one tested promoter (rsdP2) in vivo (11). However, somewhat surprisingly at the time, further experiments revealed that 6S RNA-dependent regulation of transcription is promoter specific (i.e., some promoters are downregulated in the presence of 6S RNA, while others are insensitive to 6S RNA), even during late stationary phase when Ec6S RNA levels are maximal and Eσ⁷⁰ is essentially saturated by Ec6S RNA (11, 15). Work in the last decade on 6S RNA-dependent regulation of transcription has taken two approaches: (i) study of reporter genes with minimal core promoters and mutants to reveal promoter features that determine 6S RNA sensitivity and (ii) identification of the 6S RNA regulon using global approaches.

Identification of promoter features that determine regulation by Ec6S RNA

The similarity of binding of Ec6S RNA and promoter DNA to Eσ⁷⁰ strongly suggested a direct competition model for regulation of transcription, and in vitro binding studies demonstrated that Ec6S RNA blocks binding of promoter DNA (63), as also observed in B. subtilis for both Bs6S-1 and Bs6S-2 RNAs (73). However, a set of promoters reported to respond to changes in RNAP concentrations (74) were not uniformly sensitive to Ec6S RNA, nor did 6S RNA sensitivity correlate with affinity for RNAP binding (37). Furthermore, mutagenesis of several studied promoters revealed that strength of the −35 element or the presence of an extended −10 element specifically determined 6S RNA sensitivity, and that changing these parameters alone could interconvert sensitive and insensitive promoters (37). Strength of the core −10 element did not contribute to 6S RNA sensitivity, in conflict with a model of direct competition of 6S RNA and promoter DNA for free Eσ⁷⁰. Future work will be required to provide a better understanding of the mechanistic details of 6S RNA regulation of transcription in E. coli and other bacteria.

Often, in vitro transcription assays are used to provide mechanistic insight. However, studies examining Ec6S RNA regulation of transcription in vitro, with purified components or in cell lysate, demonstrated a lack of correlation between in vitro and in vivo observations, which also was observed for Bs6S-1 and Bs6S-2 RNAs. Specifically, in vitro, all tested promoters were strongly inhibited in the presence of Ec6S RNA or Bs6S-1 or Bs6S-2 RNAs (14, 73, 75). However, in vivo, some promoters were modestly downregulated (∼2- to 5-fold) in the presence of Ec6S RNA, while others were insensitive to Ec6S RNA (15, 38, 76); similar results were observed in B. subtilis (35; Cavanagh and Wassarman, unpublished). In vitro assays in the E. coli system also exhibited a strong order-of-addition effect, and both Ec6S RNA and promoter DNA bind to Eσ⁷⁰ very tightly, which led to the hypothesis that standard in vitro transcription assays do not represent dynamic exchange that must be occurring in vivo. Whether the differences between in vivo and in vitro observations are due solely to differences in dynamics or whether other factors influence Ec6S RNA regulation in vivo that are lacking or inactive in purified and lysate systems remains to be determined.

Identification of the 6S RNA regulon in E. coli

Global expression studies revealed that many hundreds of mRNA levels were altered in cells lacking Ec6S RNA compared to wild-type cells in exponential phase, early stationary phase, and late stationary phase (37, 76). Global studies identify both those genes altered through direct 6S RNA action as well as those altered through secondary effects. Nevertheless, 6S RNA-dependent changes in late-stationary-phase genes containing mapped promoters correlated fairly well with the promoter features determined through reporter analysis (i.e., weak −35 element or extended −10 element for sensitive promoters) (37). Changes earlier in growth did not correlate well with these identified promoter features (76), which may be due to a decreased response when 6S RNA levels are not maximal, or potentially due to more-secondary effects, as several targets of Ec6S RNA regulation are regulators of transcription (e.g., Crp) or generate molecules that regulate transcription (e.g., RelA, a ppGpp synthetase) at these times (36, 37, 76). Additionally, both global studies identified genes that were increased in the presence of 6S RNA, which are likely to be secondary effects and may include 6S RNA influences on alternative σ factor utilization.

Biological role for 6S RNA function

Of note is that both direct and secondary effects of 6S RNA action are relevant for understanding the physiological consequences of regulation, as opposed to mechanistic understanding of 6S RNA function, for which only direct effects are relevant. Phenotypes associated with lack of Ec6S RNA are subtle, in part contributing to the long delay between identification of 6S RNA and the first reports of its function in regulating RNAP (9, 11). However, Ec6S RNA has been shown to contribute to competitive survival in stationary phase (time scale of days) as well as survival in long-term stationary phase when not in competitive growth (time scale of weeks) (15). Additionally, 6S RNA-dependent changes in transcription of one target gene, pspF, have been shown to alter survival at high pH in stationary phase (16), suggesting a connection between cell survival and stress response as a primary role for 6S RNA. Results from the large-scale gene expression studies similarly suggest that 6S RNA is integrated into global pathways including regulation of factors that impact transcription (e.g. Crp, FNR, and ppGpp via RelA) and general translation machinery (36, 37, 76). Detailed understanding of which gene changes are important for various phenotypes awaits future work.

6S RNA Regulation of Transcription in Diverse Bacteria

Global gene expression or proteomic studies to address the role of 6S RNA in transcription have been done in other species (e.g., B. subtilis [35; Cavanagh and Wassarman, unpublished], L. pneumophila [29], and Synechocystis 6803 [42]). Intriguingly, there have been some differences in observations from these studies and the E. coli studies (37, 76). Of note, there were many fewer mRNAs with altered levels (e.g., 135 for L. pneumophila, fewer in B. subtilis and Synechocystis) than observed in E. coli (>800). Whether this large difference represents a true difference in regulatory impact, a difference in timing of analysis, a difference between maximal 6S RNA levels and maximal binding to RNAP, or a potential difference in direct versus secondary effects remains to be tested. The other key difference was a preponderance of mRNAs that were higher in wild-type cells compared to cells lacking 6S RNA (e.g., 127 out of 135 in L. pneumophila), in contrast to E. coli, where most were decreased in wild-type compared to mutant cells, consistent with an inhibitory role for 6S RNA. However, in all cases both increased and decreased mRNAs were observed as expected when examining both direct and secondary effects. It remains an open question whether the mechanism of regulation of transcription is common for all of these reported 6S RNAs, although in all cases to date, the changes in gene expression observed suggest a biological role for 6S RNA in response to environmental conditions.

6S RNA—A TEMPLATE FOR pRNA SYNTHESIS

Perhaps one of the most exciting discoveries about Ec6S RNA in the past 15 years was that it not only structurally resembled open complex DNA but could be used as a template by RNAP to generate pRNA (63, 75), a process referred to as pRNA synthesis. When bound to 6S RNA, the DNA-dependent RNAP is converted to a specialized RNA-dependent RNAP. Detection of pRNA synthesis in vitro provided strong evidence for the similarity of Ec6S RNA and open complex DNA interactions with Eσ⁷⁰. However, it was the determination that pRNA synthesis occurs in vivo, along with subsequent work, that demonstrated that pRNA synthesis is one mechanism to regulate Ec6S RNA by contributing to the off-rate of 6S RNA from RNAP (34, 63, 77, 78). For bacteria beyond E. coli and B. subtilis, the specific role of pRNA synthesis has not been investigated directly, but pRNAs have been detected in vivo for additional bacterial species (e.g., H. pylori [57], R. sphaeroides [47], and A. aeolicus [70]), suggesting that pRNA synthesis from 6S RNAs is widespread and potentially ubiquitous.

Mechanism of pRNA Synthesis

Detailed biochemical experiments have examined mechanistic aspects of pRNA synthesis, primarily for E. coli and B. subtilis 6S RNAs (34, 63, 69, 72, 73, 75, 77, 79–81). Initiation of pRNA synthesis resembles transcription initiation in many ways, including the potential to generate abortive initiation intermediates and release of σ⁷⁰ prior to completion of RNA synthesis. However, unlike transcription, the 6S RNA-RNAP complex is not able to transition into a stable elongation complex, but instead dissociates. In fact, the 6S RNA and pRNA are released as a hybrid (6S RNA-pRNA). This released 6S RNA-pRNA hybrid is unable to rebind RNAP, thereby providing a release mechanism that cannot be reversed without further action. The fact that both regulation of transcription by 6S RNA and regulation of 6S RNA by pRNA synthesis are mediated by the positioning of 6S RNA within the active site of RNAP provides an elegantly simple mechanism to respond to environmental signals both positively and negatively.

Features of E. coli and B. subtilis 6S RNAs that contribute to mechanisms of release of 6S RNA-pRNA hybrids from RNAP also have been addressed biochemically (73, 79–81). As pRNA synthesis proceeds, the RNA central bubble is extended, which reveals additional single-stranded RNA sequence that participates in an alternative structure. This structure change facilitates the release of 6S RNA from RNAP as a 6S RNA-pRNA duplex. The details of the alternative structures are not the same for E. coli and B. subtilis 6S RNAs, but the consequences remain the same: destabilization of the interaction with RNAP, resulting in release and contributing to the length of pRNA generated and the timing of release. A. aeolicus 6S RNA forms a release structure similar to Bs6S-1 RNA (70), suggesting conservation of this release mechanism, although more information about diverse 6S RNAs is needed before strong conclusions can be made.

Differences in pRNA synthesis have been observed between E. coli and B. subtilis, most notably in preference for initiating nucleotide identity (72). Although E. coli Eσ⁷⁰ has a preference to initiate pRNA synthesis with a purine, as is also observed generally for transcription, this enzyme will initiate readily with any nucleotide in both pRNA synthesis and transcription. In contrast, B. subtilis Eσ^A demonstrates a much stronger preference for initiating pRNA synthesis with GTP over ATP, UTP, or CTP, although B. subtilis contains promoters that direct initiation with any of the four nucleotides, suggesting that this trend does not extend to transcription. Bs6S-1 RNA directs pRNA synthesis initiation with GTP, while Bs6S-2 RNA directs initiation with ATP, leading to a large difference in efficiency of pRNA synthesis, at least as measured in vitro (72). A similar preference for initiation with GTP by B. subtilis Eσ^A was observed in vitro and in vivo on E. coli 6S RNA, which directs initiation with ATP, compared to a mutant E. coli 6S RNA, 6S (iGTP) RNA, that directs initiation with GTP (72). Thus, the difference in preference for initiating nucleotide in pRNA synthesis between E. coli and B. subtilis originates from differences in RNAP, although the mechanistic details await further study. The impact of the observed difference in pRNA efficiency in vitro on in vivo function and regulation of Bs6S-1 and Bs6S-2 RNAs is likely to continue to be an area of active research (34, 82).

Biological Role for pRNA Synthesis

A burst of pRNA synthesis from Ec6S RNA and Bs6S-1 RNA occurs in vivo within minutes of diluting stationary-phase cells into fresh medium (outgrowth) (34, 63, 69, 77), suggesting a role for this process in the transition out of stationary phase to allow restart of growth. The released 6S RNA remains base-paired to the pRNA, thus preventing rebinding to Eσ⁷⁰ or Eσ^A due to the altered structure (34, 63, 73, 77, 79). The majority (>90%) of Ec6S and Bs6S-1 RNAs are degraded during outgrowth, presumably as a consequence of release from RNAP, suggesting that pRNA synthesis may play an important role in determining Ec6S RNA and Bs6S-1 RNA accumulation profiles. Ec6S- and Bs6S-1-directed pRNA levels were not detected in late stationary phase in one study (34), supporting a hypothesis that pRNA synthesis initiation is sensitive to nucleotide triphosphate levels, linking pRNA synthesis timing to outgrowth. The influence of nucleotide levels on pRNA synthesis efficiency has been noted in vitro (34, 63, 73, 77, 79). However, other studies have detected pRNA from Bs6S-1 RNA (and Bs6S-2 RNA) in both exponential and earlier stationary phase (82), although timing in stationary phase, strains, and growth conditions varied between different studies. Changes in pRNA length at different stages of growth also have been observed (69), suggesting the potential for additional levels of regulation of pRNA synthesis, such as in pRNA synthesis elongation and release rates in response to nucleotide levels. Certainly further study of these different observations and the potential connections between pRNA synthesis, 6S RNA stability and accumulation profiles, and the impact of pRNA length distribution need to be addressed experimentally in all organisms. More quantitative information about pRNA synthesis efficiency throughout growth is needed, and an examination of whether additional cellular signals or factors regulate pRNA synthesis is required before the contribution of pRNA synthesis in regulating 6S RNA levels throughout growth is fully understood.

The impact of pRNA synthesis on the ability of cells to restart growth upon exit from stationary phase (i.e., outgrowth) was investigated in E. coli using mutant Ec6S RNAs that retain the ability to bind Eσ⁷⁰ but lack the ability to be released from Eσ⁷⁰ through pRNA synthesis (34, 78). In one study, it was found that cells expressing a mutant 6S RNA were delayed in restarting growth, suggesting that pRNA synthesis-mediated release of Eσ⁷⁰ from Ec6S RNA is required for efficient restart of growth after stationary phase (34). Toxic effects of expressing this type of mutant RNA also were observed, although the extent of toxicity was dependent on expression levels (34, 78). However, the nonreleasing 6S RNA mutant regulated transcription similarly to wild-type 6S RNA in stationary phase, both in specificity of promoters sensitive and insensitive to 6S RNA and the extent of regulation (34). Therefore, it has been suggested that pRNA synthesis is one mechanism to regulate 6S RNA accumulation profiles and to facilitate concerted release of Eσ⁷⁰ during outgrowth but does not otherwise influence mechanisms of 6S RNA action in regulating transcription. Although it is enticing to question whether pRNA itself has a function as an sRNA in addition to the role of its synthesis in regulating 6S RNA levels, currently there is no evidence to support an independent function, and no mutant phenotypes have been revealed in cells expressing mutant Ec6S RNAs that direct synthesis of pRNAs with different sequences (Cavanagh and Wassarman, unpublished). In B. subtilis, a requirement for pRNA synthesis-mediated release of Eσ^A to promote efficient outgrowth has also been demonstrated (34).

6S RNA—FUTURE QUESTIONS

Many questions about 6S RNA function in E. coli and to a lesser extent in B. subtilis have been addressed at some level. However, even in these well-studied organisms questions remain, including how specific changes in gene expression contribute to phenotypes associated with loss of 6S RNA function and what specific mechanisms mediate promoter-specific regulation of transcription. Even more questions await investigation in other bacteria and include the following. (i) How many genes are regulated by 6S RNA, and by what mechanism(s)? (ii) How are diverse 6S RNA structures recognized by their cognate RNAPs, and which interactions are conserved or species specific? (iii) What are the details underpinning the relationship between accumulation profile and physiological impact? (iv) What other factors contribute to regulation of 6S RNA activity beyond pRNA synthesis? (v) Are there broader impacts of pRNA synthesis beyond the studied release mechanism?