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.

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.

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.

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 Here we identified a novel mechanism in which CsrA represses translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and fusions demonstrated that CsrA represses expression.

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 P 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.

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).

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.

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.

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.

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.

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.