Abstract: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 … Show more
“…The differences in accumulation of Bs 6S-1 and Bs 6S-2 RNAs or Lp 6S and Lp 6S2 RNAs suggest that they likely have different physiological roles, in agreement with observations that gene expression and proteomic profiles for cells lacking Bs 6S-1 and Bs 6S-2 RNAs are different (33, 36), and observed mutant phenotypes (i.e. altered timing of sporulation and cell density changes in stationary phase) are associated specifically with the loss of Bs 6S-1 RNA (34, 36). Streptomyces coelicolor 6S RNA also accumulates with a profile similar to Ec 6S RNA and influences growth rate (41).…”
Section: S Rnas Are Widespread Throughout the Bacterial Kingdomsupporting
6S RNA is a small RNA regulator of RNA polymerase that is present broadly throughout the bacterial kingdom. Initial functional studies in Escherichia coli revealed that 6S RNA forms a complex with RNA polymerase 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: 1. Addressing questions of how broadly conserved 6S RNAs are in diverse organisms through continued identification and initial characterization of divergent 6S RNAs; 2. The nature of the 6S RNA-RNA polymerase interaction through examination of variant proteins and mutant RNAs, crosslinking approaches, and ultimately a cryo-EM structure; 3. The physiological consequences of 6S RNA function through identification of the 6S RNA-regulon and promoter features that determine 6S RNA sensitivity; 4. The mechanism and cellular impact of 6S RNA-directed synthesis of pRNAs (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.
“…The differences in accumulation of Bs 6S-1 and Bs 6S-2 RNAs or Lp 6S and Lp 6S2 RNAs suggest that they likely have different physiological roles, in agreement with observations that gene expression and proteomic profiles for cells lacking Bs 6S-1 and Bs 6S-2 RNAs are different (33, 36), and observed mutant phenotypes (i.e. altered timing of sporulation and cell density changes in stationary phase) are associated specifically with the loss of Bs 6S-1 RNA (34, 36). Streptomyces coelicolor 6S RNA also accumulates with a profile similar to Ec 6S RNA and influences growth rate (41).…”
Section: S Rnas Are Widespread Throughout the Bacterial Kingdomsupporting
6S RNA is a small RNA regulator of RNA polymerase that is present broadly throughout the bacterial kingdom. Initial functional studies in Escherichia coli revealed that 6S RNA forms a complex with RNA polymerase 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: 1. Addressing questions of how broadly conserved 6S RNAs are in diverse organisms through continued identification and initial characterization of divergent 6S RNAs; 2. The nature of the 6S RNA-RNA polymerase interaction through examination of variant proteins and mutant RNAs, crosslinking approaches, and ultimately a cryo-EM structure; 3. The physiological consequences of 6S RNA function through identification of the 6S RNA-regulon and promoter features that determine 6S RNA sensitivity; 4. The mechanism and cellular impact of 6S RNA-directed synthesis of pRNAs (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.
“…However, this difference in behavior is probably not the only difference in activity between these two RNAs. Cells lacking 6S-1 RNA also exhibit an early sporulation phenotype, which has been postulated to result from a more rapid depletion of nutrients by these cells compared with wild-type cells (19). However, cells lacking 6S-2 RNA sporulate with normal timing, and it is difficult to imagine how changes in pRNA synthesis rates would alter cell behavior after entry into stationary phase when pRNA synthesis of 6S-1 RNA already is low (7,16).…”
Section: Discussionmentioning
confidence: 99%
“…Other observations suggest differential use for pRNA synthesis is unlikely to be the only difference in behavior between these two RNAs. For example, cells lacking Bs6S-1 initiate sporulation earlier than wild-type cells or cells lacking Bs6S-2 RNA, a time when pRNA synthesis rates are low (19). Figure 1.The efficiency of pRNA synthesis from Bs6S-1 RNA and Bs6S-2 RNA is dependent on sequences in the central bulge.…”
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.
“…Among them, one of the most widespread and abundant (approximately 10,000 copies per cells in stationary phase) is the 6S RNA, a global regulator sRNA that reduces the expression of several σ-70 dependent promoters, favoring the interaction of RNA polymerase with alternate sigma factors, such as RpoS in Bacillus subtilis (Cavanagh and Wassarman 2014), and has been implicated in the down-regulation of the expression of key pathways in response to changing stressful conditions and growth adaptation (Cavanagh et al 2010;Cavanagh and Wassarman 2013). GcvB is also one of the most highly conserved Hfq-associated sRNAs in Gram-negative bacteria and was previously reported to regulate many genes involved in the transport and biosynthesis of oligopeptides and amino acids, such as the branched-chain amino acid (BCAA) transport system (Sharma et al 2011;Stauffer and Stauffer 2013).…”
Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies.
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