Regulation of RyeA/SraC expression in Escherichia coli

BMC Genomics, 2014

Nucleic Acids and Molecular Biology, 2006

In recent years, small regulatory RNAs have been discovered at a staggering rate both in prokaryotes and eukaryotes. By now it is clear that post-transcriptional regulation of gene expression mediated by such RNAs is the rule rather than-as previously believed-the exception. In this chapter, we focus on small RNAs (sRNAs) encoded by bacterial chromosomes. The strategies for their discovery, their biological roles, and their mechanisms of action are discussed. Even though the number of well-characterized sR-NAs in, for example, the best studied model enterobacterium Escherichia coli,i ss t i l l small, the emerging pattern suggests that antisense mechanisms predominate. In terms of their roles in bacterial physiology, most of these RNAs appear to be involved in stress response regulation. Some other examples indicate functions in regulation of virulence. Two aspects of sRNA-mediated control arising from recent observations are addressed as well. Firstly, some sRNAs need proteins (notably Hfq) as helpers in their antisense activities-at this point the reason for this requirement is not understood. Secondly, only limited sequence complementarity is generally observed in antisense-target RNA pairs. This raises the fundamental question of how specific recognition is accomplished, and what the structure/sequence determinants for rapid and productive interaction are. 2 E.G.H. Wagner · F. Darfeuille

Journal of Bacteriology, 2007

RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia coli . Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB , negatively regulated the fusion. An insertion in the rep helicase and one upstream of dnaK decreased expression of the fusion. Multicopy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the gene for the response regulator OmpR; the second contained mipA , encoding a murein hydrolase. The involvement of MipA and OmpR in cell surface synthesis suggested that the rybB promoter might be dependent on σ E . The sequence upstream of the +1 of rybB contains a consensus σ E promoter. The activity of rybB - lacZ was increased in cells lacking the RseA anti-sigma factor and when σ E was overproduced from ...

Journal of Biological Chemistry, 2010

Small non-coding RNAs (sRNA) have emerged as important elements of gene regulatory circuits. In enterobacteria such as Escherichia coli and Salmonella many of these sRNAs interact with the Hfq protein, an RNA chaperone similar to mammalian Sm-like proteins and act in the post-transcriptional regulation of many genes. A number of these highly conserved ribo-regulators are stringently regulated at the level of transcription and are part of major regulons that deal with the immediate response to various stress conditions, indicating that every major transcription factor may control the expression of at least one sRNA regulator. Here, we extend this view by the identification and characterization of a highly conserved, anaerobically induced small sRNA in E. coli, whose expression is strictly dependent on the anaerobic transcriptional fumarate and nitrate reductase regulator (FNR). The sRNA, named FnrS, possesses signatures of base-pairing RNAs, and we show by employing global proteomic and transcriptomic profiling that the expression of multiple genes is negatively regulated by the sRNA. Intriguingly, many of these genes encode enzymes with "aerobic" functions or enzymes linked to oxidative stress. Furthermore, in previous work most of the potential target genes have been shown to be repressed by FNR through an undetermined mechanism. Collectively, our results provide insight into the mechanism by which FNR negatively regulates genes such as sodA, sodB, cydDC, and metE, thereby demonstrating that adaptation to anaerobic growth involves the action of a small regulatory RNA.

Journal of Bacteriology, 2009

The Escherichia coli RNA degradosome is a protein complex that plays a critical role in the turnover of numerous RNAs. The key component of the degradosome complex is the endoribonuclease RNase E, a multidomain protein composed of an N-terminal catalytic region and a C-terminal region that organizes the other protein components of the degradosome. Previously, the RNase E inhibitors RraA and RraB were identified genetically and shown to bind to the C-terminal region of RNase E, thus affecting both the protein composition of the degradosome and the endonucleolytic activity of RNase E. In the present work, we investigated the transcriptional regulation of rraB . rraB was shown to be transcribed constitutively from its own promoter, P rraB . Transposon mutagenesis and screening for increased β-galactosidase activity from a chromosomal P rraB-lacZ transcriptional fusion resulted in the isolation of a transposon insertion in glmS , encoding the essential enzyme glucosamine-6-phosphate syn...

Frontiers in Molecular Biosciences, 2020

Small non-coding RNAs (sRNAs) are critical post-transcriptional regulators of gene expression. Distinct RNA-binding proteins (RBPs) influence the processing, stability and activity of bacterial small RNAs. The vast majority of bacterial sRNAs interact with mRNA targets, affecting mRNA stability and/or its translation rate. The assistance of RNA-binding proteins facilitates and brings accuracy to sRNA-mRNA basepairing and the RNA chaperones Hfq and ProQ are now recognized as the most prominent RNA matchmakers in bacteria. These RBPs exhibit distinct high affinity RNA-binding surfaces, promoting RNA strand interaction between a trans-encoding sRNA and its mRNA target. Nevertheless, some organisms lack ProQ and/or Hfq homologs, suggesting the existence of other RBPs involved in sRNA function. Along this line of thought, the global regulator CsrA was recently shown to facilitate the access of an sRNA to its target mRNA and may represent an additional factor involved in sRNA function. Ribonucleases (RNases) can be considered a class of RNA-binding proteins with nucleolytic activity that are responsible for RNA maturation and/or degradation. Presently RNase E, RNase III, and PNPase appear to be the main players not only in sRNA turnover but also in sRNA processing. Here we review the current knowledge on the most important bacterial RNA-binding proteins affecting sRNA activity and sRNA-mediated networks.

2004

Evidence is accumulating that small, noncoding RNAs are important regulatory molecules. Computational and experimental searches have led to the identification of 60 small RNA genes in Escherichia coli. However, most of these studies focused on the intergenic regions and assumed that small RNAs were.50 nt. Thus, the previous screens missed small RNAs encoded on the antisense strand of protein-coding genes and small RNAs of,50 nt. To identify additional small RNAs, we carried out a cloning-based screen focused on RNAs of 30–65 nt. In this screen, we identified RNA species corresponding to fragments of rRNAs, tRNAs and known small RNAs. Several of the small RNAs also corresponded to 50- and 30-untranslated regions (UTRs) and internal fragments of mRNAs. Four of the 30-UTR-derived RNAs were highly abundant and two showed expression patterns that differed from the corresponding mRNAs, suggesting independent functions for the 30-UTR-derived small RNAs. We also detected three previously un...

Nucleic Acids Research, 2008

The DNA damage induced SOS response in Escherichia coli is initiated by cleavage of the LexA repressor through activation of RecA. Here we demonstrate that overexpression of the SOSinducible tisAB gene inhibits several SOS functions in vivo. Wild-type E. coli overexpressing tisAB showed the same UV sensitivity as a lexA mutant carrying a noncleavable version of the LexA protein unable to induce the SOS response. Immunoblotting confirmed that tisAB overexpression leads to higher levels of LexA repressor and northern experiments demonstrated delayed and reduced induction of recA mRNA. In addition, induction of prophage j and UV-induced filamentation was inhibited by tisAB overexpression. The tisAB gene contains antisense sequences to the SOS-inducible dinD gene (16 nt) and the uxaA gene (20 nt), the latter encoding a dehydratase essential for galacturonate catabolism. Cleavage of uxaA mRNA at the antisense sequence was dependent on tisAB RNA expression. We showed that overexpression of tisAB is less able to confer UV sensitivity to the uxaA dinD double mutant as compared to wild-type, indicating that the dinD and uxaA transcripts modulate the anti-SOS response of tisAB. These data shed new light on the complexity of SOS regulation in which the uxaA gene could link sugar metabolism to the SOS response via antisense regulation of the tisAB gene.

Cold Spring Harbor perspectives in biology, 2011

Small RNA regulators (sRNAs) have been identified in a wide range of bacteria and found to play critical regulatory roles in many processes. The major families of sRNAs include true antisense RNAs, synthesized from the strand complementary to the mRNA they regulate, sRNAs that also act by pairing but have limited complementarity with their targets, and sRNAs that regulate proteins by binding to and affecting protein activity. The sRNAs with limited complementarity are akin to eukaryotic microRNAs in their ability to modulate the activity and stability of multiple mRNAs. In many bacterial species, the RNA chaperone Hfq is required to promote pairing between these sRNAs and their target mRNAs. Understanding the evolution of regulatory sRNAs remains a challenge; sRNA genes show evidence of duplication and horizontal transfer but also could be evolved from tRNAs, mRNAs or random transcription.

Nucleic Acids Research, 2003

Small RNA (sRNA) molecules have gained much interest lately, as recent genome-wide studies have shown that they are widespread in a variety of organisms. The relatively small family of 10 known sRNA-encoding genes in Escherichia coli has been signi®cantly expanded during the past two years with the discovery of 45 novel genes. Most of these genes are still uncharacterized and their cellular roles are unknown. In this survey we examined the sequence and genomic features of the 55 currently known sRNA-encoding genes in E.coli, attempting to identify their common characteristics. Such characterization is important for both expanding our understanding of this unique gene family and for improving the methods to predict and identify sRNA-encoding genes based on genomic information.

FEMS Microbiology Letters, 2015

Bacterial regulatory networks of gene expression include the interaction of diverse types of molecules such as the small non-coding RNAs (sRNAs) and their cognate messenger RNAs (mRNAs). In this study, we demonstrated that the Salmonella Typhimurium sRNA SroC is significantly expressed between late-exponential and stationary phase of growth in an rpoS-dependent manner. The expression of flagellar genes predicted as targets of this sRNA was quantitatively analyzed in both, a ΔsroC mutant and a SroC-overexpressing (pSroC) strain. Deletion of sroC increased flagellar gene-expression (i.e., flhBAE and fliE). Conversely, overexpression of SroC reduced flhBAE and fliE expression. These observations correlated with phenotypic evaluation of motility, where sroC deletion slightly increased motility, which in turn, was drastically reduced upon overexpression of SroC. The effects of deletion and overexpression of sroC in biofilm formation were also examined, where the ΔsroC and pSroC strains exhibited a reduced and increased ability to form biofilm, respectively. Furthermore, an electron microscopy revealed that wild-type strain overexpressing SroC had a non-flagellated phenotype. Taken together, our results showed that S. Typhimurium sRNA SroC modulates the flagellar synthesis by downregulating the expression of flhBAE and fliE genes.

Scientific Reports, 2015

In bacteria, selective promoter recognition by RNA polymerase is achieved by its association with σ factors, accessory subunits able to direct RNA polymerase "core enzyme" (E) to different promoter sequences. Using Chromatin Immunoprecipitation-sequencing (ChIP-seq), we searched for promoters bound by the σ S -associated RNA polymerase form (Eσ S ) during transition from exponential to stationary phase. We identified 63 binding sites for Eσ S overlapping known or putative promoters, often located upstream of genes (encoding either ORFs or non-coding RNAs) showing at least some degree of dependence on the σ S -encoding rpoS gene. Eσ S binding did not always correlate with an increase in transcription level, suggesting that, at some σ S -dependent promoters, Eσ S might remain poised in a pre-initiation state upon binding. A large fraction of Eσ S -binding sites corresponded to promoters recognized by RNA polymerase associated with σ 70 or other σ factors, suggesting a considerable overlap in promoter recognition between different forms of RNA polymerase. In particular, Eσ S appears to contribute significantly to transcription of genes encoding proteins involved in LPS biosynthesis and in cell surface composition. Finally, our results highlight a direct role of Eσ S in the regulation of non coding RNAs, such as OmrA/B, RyeA/B and SibC.

RNA Biology, 2012

Physiological implications and possible regulatory mechanisms are discussed. Results Characterization of RybA. Wassarman et al. tested the expression of RybA under three conditions: LB-exponential phase, LB-stationary phase and minimal medium. 15 We analyzed the expression pattern of RybA under more conditions in E. coli W3110. To this end, total RNA samples were isolated from cells grown under ten different conditions (see Materials and Methods) and subjected to northern analysis (Fig. 1A). As already described in reference 15, RybA is present in at least two forms of roughly 300 and 200 nt. RybA is detectable under all conditions tested. Under cold shock, peroxide stress and, to a lesser extent, under superoxide stress the expression of the sRNA is strongly upregulated. This result indicates that RybA might play a role during cold shock and/or oxidative stress. In this study we concentrated our investigations on the role of RybA under peroxide stress. The 5' and 3' ends of RybA were not known prior to this study. We determined the boundaries of RybA during the exponential phase and under peroxide stress by primer extension analysis and 5' and 3' RACE (Figs. 1B; Fig. S1). The predominant forms of RybA are 323 and 207 nt long, respectively. 5' RACE after treatment of total RNA with Tobacco Acid Pyrophosphatase (TAP) did not produce any additional band in comparison with untreated RNA. We therefore could not detect the primary rybA-transcript by 5' RACE (Fig. S1B). It is probably too short-lived to be detectable. However this experiment demonstrates that the +1 band detected is indeed the result of some RNA maturation. Northern analysis revealed that neither RNase III nor the degradosome-bound RNase E had an influence on RybA accumulation and maturation during peroxide stress (Fig. S2). However, we cannot exclude that RNase E alone might play a role in RybA homeostasis since the RNase E mutant used during this experiment was only defective for degradosome inclusion of the protein, which retained full enzymatic activity. The sequence coordinates of RybA in the E. coli genome are indicated in Figure 1C. A predicted σ 70 promoter is located directly upstream of the 5' end of RybA and contains a-35 box (TTA ACG) and a-10 box (TATAAT) starting at nucleotides 852,321 and 852,300 respectively (Fig. S3C). However, this predicted promoter is 17 nucleotides too far upstream from the first nucleotide of RybA as determined by primer extension analysis and 5' RACE. We also performed a primer extension analysis with pRybA (pKG09, Table 1) a multicopy plasmid carrying the intergenic region containing RybA between the

BMC Genomics, 2011

Background In Escherichia coli, approximately 100 regulatory small RNAs (sRNAs) have been identified experimentally and many more have been predicted by various methods. To provide a comprehensive overview of sRNAs, we analysed the low-molecular-weight RNAs (< 200 nt) of E. coli with deep sequencing, because the regulatory RNAs in bacteria are usually 50-200 nt in length. Results We discovered 229 novel candidate sRNAs (≥ 50 nt) with computational or experimental evidence of transcription initiation. Among them, the expression of seven intergenic sRNAs and three cis-antisense sRNAs was detected by northern blot analysis. Interestingly, five novel sRNAs are expressed from prophage regions and we note that these sRNAs have several specific characteristics. Furthermore, we conducted an evolutionary conservation analysis of the candidate sRNAs and summarised the data among closely related bacterial strains. Conclusions This comprehensive screen for E. coli sRNAs using a deep sequenci...

Nucleic Acids Research, 2017

Small RNAs are key components of complex regulatory networks. These molecules can integrate multiple cellular signals to control specific target mR-NAs. The recent development of high-throughput methods tremendously helped to characterize the full targetome of sRNAs. Using MS2-affinity purification coupled with RNA sequencing (MAPS) technology, we reveal the targetomes of two sRNAs, CyaR and RprA. Interestingly, both CyaR and RprA interact with the 5-UTR of hdeD mRNA, which encodes an acidresistance membrane protein. We demonstrate that CyaR classically binds to the RBS of hdeD, interfering with translational initiation. We identified an A/Urich motif on hdeD, which is bound by the RNA chaperone Hfq. Our results indicate that binding of this motif by Hfq is required for CyaR-induced degradation of hdeD mRNA. Additional data suggest that two molecules of RprA must bind the 5-UTR of hdeD to block translation initiation. Surprisingly, while both CyaR and RprA sRNAs bind to the same motif on hdeD mRNA, RprA solely acts at the translational level, leaving the target RNA intact. By interchanging the seed region of CyaR and RprA sRNAs, we also swap their regulatory behavior. These results suggest that slight changes in the seed region could modulate the regulation of target mRNAs.