Coordinate regulation of the expression of SdsR toxin and its downstream pphA gene by RyeA antitoxin in Escherichia coli.

In Escherichia coli, SdsR and RyeA, a unique pair of mutually cis-encoded small RNAs (sRNAs), act as toxin and antitoxin, respectively. SdsR and RyeA expression are reciprocally regulated; however, how each regulates the synthesis of the other remains unclear. Here, we characterized the biosynthesis of the two sRNAs during growth and investigated their coordinate regulation using sdsR and ryeA promoter mutant strains. We found that RyeA transcription occurred even upon entry of cells into the stationary phase, but its apparent expression was restricted to exponentially growing cells because of its degradation by SdsR. Likewise, the appearance of SdsR was delayed owing to its RyeA-mediated degradation. We also found that the sdsR promoter was primarily responsible for transcription of the downstream pphA gene encoding a phosphatase and that pphA mRNA was synthesized by transcriptional read-through over the sdsR terminator. Transcription from the σ70-dependent ryeA promoter inhibited transcription from the σS-dependent sdsR promoter through transcriptional interference. This transcriptional inhibition also downregulated pphA expression, but RyeA itself did not downregulate pphA expression.

The small RNA, SdsR, acts as a novel type of toxin in Escherichia coli.

Most small noncoding RNAs (sRNAs) are known to base pair with target mRNAs and regulate mRNA stability or translation to trigger various changes in the cell metabolism of Escherichia coli. The SdsR sRNA is expressed specifically during the stationary phase and represses tolC and mutS expression. However, it was not previously known whether the growth-phase-dependent regulation of SdsR is important for cell growth. Here, we ectopically expressed SdsR during the exponential phase and examined cell growth and survival. We found that ectopic expression of SdsR led to a significant and Hfq-dependent cell death with accompanying cell filamentation. This SdsR-driven cell death was alleviated by overexpression of RyeA, an sRNA transcribed on the opposite DNA strand, suggesting that SdsR/RyeA is a novel type of toxin-antitoxin (T/A) system in which both the toxin and the antitoxin are sRNAs. We defined the minimal region required for the SdsR-driven cell death. We also performed RNA-seq analysis and identified 209 genes whose expression levels were altered by more than two-fold following pulse expression of ectopic SdsR at exponential phase. Finally, we found that that the observed SdsR-driven cell death was mainly caused by the SdsR-mediated repression of yhcB, which encodes an inner membrane protein.

Glyoxals as in vivo RNA structural probes of guanine base-pairing.

Elucidation of the folded structures that RNA forms in vivo is vital to understanding its functions. Chemical reagents that modify the Watson-Crick (WC) face of unprotected nucleobases are particularly useful in structure elucidation. Dimethyl sulfate penetrates cell membranes and informs on RNA base-pairing and secondary structure but only modifies the WC face of adenines and cytosines. We present glyoxal, methylglyoxal, and phenylglyoxal as potent in vivo reagents that target the WC face of guanines as well as cytosines and adenines. Tests on rice (Oryza sativa) 5.8S rRNA in vitro read out by reverse transcription and gel electrophoresis demonstrate specific modification of almost all guanines in a time- and pH-dependent manner. Subsequent in vivo tests on rice, a eukaryote, and Bacillus subtilis and Escherichia coli, Gram-positive and Gram-negative bacteria, respectively, showed that all three reagents enter living cells without prior membrane permeabilization or pH adjustment of the surrounding media and specifically modify solvent-exposed guanine, cytosine, and adenine residues.

Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame.

CsrA is a global regulatory RNA binding protein that has important roles in regulating carbon metabolism, motility, biofilm formation, and numerous other cellular processes. IraD functions as an antiadapter protein that inhibits RssB-mediated degradation of RpoS, the general stress response and stationary-phase sigma factor of Escherichia coli Here we identified a novel mechanism in which CsrA represses iraD translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and lacZ fusions demonstrated that CsrA represses iraD expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the iraD leader transcript, all of which are far upstream of the iraD ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the iraD Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the iraD leader region. Two of these ORFs do not affect iraD expression. However, the translation initiation region of the third ORF contains three of the CsrA binding sites, one of which overlaps its SD sequence. Furthermore, the ORF stop codon overlaps the iraD start codon, a sequence arrangement indicative of translational coupling. In vivo expression and in vitro translation studies with wild-type and mutant reporter fusions demonstrated that bound CsrA directly represses translation initiation of this ORF. We further established that CsrA-dependent repression of iraD translation occurs entirely via translational coupling with this ORF, leading to accelerated iraD mRNA decay.IMPORTANCE CsrA posttranscriptionally represses gene expression associated with stationary-phase bacterial growth, often in opposition to the transcriptional effects of the stationary-phase sigma factor RpoS. We show that CsrA employs a novel regulatory mechanism to repress translation of iraD, which encodes an antiadapter protein that protects RpoS against proteolysis. CsrA binds to four sites in the iraD leader transcript but does not directly occlude ribosome binding to the iraD SD sequence. Instead, CsrA represses translation of a short open reading frame encoded upstream of iraD, causing repression of iraD translation via translational coupling. This finding offers a novel mechanism of gene regulation by the global regulator CsrA, and since RpoS can activate csrA transcription, this also highlights a new negative-feedback loop within the complex Csr and RpoS circuitry.

Eliminating blurry bands in gels with a simple cost-effective repair to the gel cassette.

Gel electrophoresis and subsequent imaging using phosphorimagers is one of the most important and widely used techniques in RNA and DNA analysis. Radiolabeling nucleic acids with 32P and detecting bands using a phoshorimager are useful both in a qualitative sense for nucleic acid detection and in a quantitative sense for structural, kinetic, or binding-based assays. Because of this, good resolution of gel bands based on molecular weight and size of RNA or DNA is essential for analysis. The appearance of blurry gel bands of 32P-labeled RNA and DNA thus represents a serious problem in the laboratory. A quick search on the Internet uncovers numerous reports begrudging the appearance of blurry bands, as well as attempts to fix them without success. Indeed, our laboratories were beset by the intermittent problem of blurry gels for over one year before we found a solution. Herein we describe a simple and cost-effective solution to a problem that we show originates from the phosphorimager cassettes rather than the integrity of the gel itself. We hope that the information provided here will lead to immediate help for other laboratories experiencing similar issues with labeled nucleic acid gel-based assays. The improvement in the clarity of the gels is nothing short of astonishing in many instances and will lead to higher resolution images for analysis and publications.

CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of pnp Transcripts That Have Been Previously Processed by RNase III and PNPase.

UNLABELLED: Csr is a conserved global regulatory system that represses or activates gene expression posttranscriptionally. CsrA of Escherichia coli is a homodimeric RNA binding protein that regulates transcription elongation, translation initiation, and mRNA stability by binding to the 5' untranslated leader or initial coding sequence of target transcripts. pnp mRNA, encoding the 3' to 5' exoribonuclease polynucleotide phosphorylase (PNPase), was previously identified as a CsrA target by transcriptome sequencing (RNA-seq). Previous studies also showed that RNase III and PNPase participate in a pnp autoregulatory mechanism in which RNase III cleavage of the untranslated leader, followed by PNPase degradation of the resulting 5' fragment, leads to pnp repression by an undefined translational repression mechanism. Here we demonstrate that CsrA binds to two sites in pnp leader RNA but only after the transcript is fully processed by RNase III and PNPase. In the absence of processing, both of the binding sites are sequestered in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer bridges the upstream high-affinity site to the downstream site that overlaps the pnp Shine-Dalgarno sequence such that bound CsrA causes strong repression of pnp translation. CsrA-mediated translational repression also leads to a small increase in the pnp mRNA decay rate. Although CsrA has been shown to regulate translation and mRNA stability of numerous genes in a variety of organisms, this is the first example in which prior mRNA processing is required for CsrA-mediated regulation. IMPORTANCE: CsrA protein represses translation of numerous mRNA targets, typically by binding to multiple sites in the untranslated leader region preceding the coding sequence. We found that CsrA represses translation of pnp by binding to two sites in the pnp leader transcript but only after it is processed by RNase III and PNPase. Processing by these two ribonucleases alters the mRNA secondary structure such that it becomes accessible to the ribosome for translation as well as to CsrA. As one of the CsrA binding sites overlaps the pnp ribosome binding site, bound CsrA prevents ribosome binding. This is the first example in which regulation by CsrA requires prior mRNA processing and should link pnp expression to conditions affecting CsrA activity.

Effects of different target sites on antisense RNA-mediated regulation of gene expression.

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability.

Regulation of transcription from two ssrS promoters in 6S RNA biogenesis.

ssrS-encoded 6S RNA is an abundant noncoding RNA that binds σ(70)-RNA polymerase and regulates expression at a subset of promoters in Escherichia coli. It is transcribed from two tandem promoters, ssrS P1 and ssrS P2. Regulation of transcription from two ssrS promoters in 6S RNA biogenesis was examined. Both P1 and P2 were growth phase-dependently regulated. Depletion of 6S RNA had no effect on growth-phase-dependent transcription from either promoter, whereas overexpression of 6S RNA increased P1 transcription and decreased P2 transcription, suggesting that transcription from P1 and P2 is subject to feedback activation and feedback inhibition, respectively. This feedback regulation disappeared in Δfis strains, supporting involvement of Fis in this process. The differential feedback regulation may provide a means for maintaining appropriate cellular concentrations of 6S RNA.

Exploring sRNA-mediated gene silencing mechanisms using artificial small RNAs derived from a natural RNA scaffold in Escherichia coli.

An artificial small RNA (afsRNA) scaffold was designed from an Escherichia coli sRNA, SibC. Using the lacZ reporter system, the gene silencing effects of afsRNAs were examined to explore the sRNA-mediated gene-silencing mechanisms in E. coli. Substitution of the original target recognition sequence with a new sequence recognizing lacZ mRNA led to effective reduction of lacZ gene expression. Single-strandedness of the target recognition sequences in the scaffold was essential for effective gene silencing. The target recognition sequence was shortened to 10 nt without significant loss of gene silencing, although this minimal length was limited to a specific target mRNA sequence. In cases where afsRNAs had mismatched (forming internal loops) or unmatched (forming bulges) regions in the middle of the target recognition sequence, internal loop-forming afsRNAs were more effective in gene silencing than those that formed bulges. Unexpectedly, gene silencing by afsRNA was not decreased but increased on hfq disruption in E. coli, particularly when interactions between afsRNA and mRNA were weak, suggesting that Hfq is possibly involved in destabilization of the RNA-RNA duplex, rather than enhancement of base pairing.

Rho-dependent termination of ssrS (6S RNA) transcription in Escherichia coli: implication for 3' processing of 6S RNA and expression of downstream ygfA (putative 5-formyl-tetrahydrofolate cyclo-ligase).

It is well known that 6S RNA, a global regulatory noncoding RNA that modulates gene expression in response to the cellular stresses in Escherichia coli, is generated by processing from primary ssrS (6S RNA) transcripts derived from two different promoters. The 5' processing of 6S RNA from primary transcripts has been well studied; however, it remains unclear how the 3'-end of this RNA is generated although previous studies have suggested that exoribonucleolytic trimming is necessary for 3' processing. Here, we describe several Rho-dependent termination sites located ∼90 bases downstream of the mature 3'-end of 6S RNA. Our data suggest that the 3'-end of 6S RNA is generated via exoribonucleolytic trimming, rather than endoribonucleolytic cleavage, following the transcription termination events. The termination sites identified in this study are within the open reading frame of the downstream ygfA (putative 5-formyl-tetrahydrofolate cyclo-ligase) gene, a part of the highly conserved bacterial operon ssrS-ygfA, which is up-regulated during the biofilm formation. Our findings reveal that ygfA expression, which also aids the formation of multidrug-tolerant persister cells, could be regulated by Rho-dependent termination activity in the cell.

Recognition and discrimination of target mRNAs by Sib RNAs, a cis-encoded sRNA family.

Five Sib antitoxin RNAs, members of a family of cis-encoded small regulatory RNAs (sRNAs) in Escherichia coli, repress their target mRNAs, which encode Ibs toxins. This target repression occurs only between cognate sRNA-mRNA pairs with an exception of ibsA. We performed co-transformation assays to assess the ability of SibC derivatives to repress ibsC expression, thereby revealing the regions of SibC that are essential for ibsC mRNA recognition. SibC has two target recognition domains, TRD1 and TRD2, which function independently. The target site for TRD1 is located within the ORF of ibsC, whereas the target site for TRD2 is located in the translational initiation region. The TRD1 sequence is sufficient to repress ibsC expression. In contrast, TRD2 requires a specific structure in addition to the recognition sequence. An in vitro structural probing analysis showed that the initial interactions at these two recognition sites allowed base-pairing to progress into the flanking sequences. Displacement of the TRD1 and TRD2 domains of SibC by the corresponding domains of SibD changed the target specificity of SibC from ibsC to ibsD, suggesting that these two elements modulate the cognate target recognition of each Sib RNA by discriminating among non-cognate ibs mRNAs.