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      A novel sRNA component of the carbon storage regulatory system of Escherichia coli.

      Molecular Microbiology
      Base Sequence, Biofilms, Carbon, metabolism, Escherichia coli, genetics, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Bacterial, Glycogen, Molecular Sequence Data, Nucleic Acid Conformation, RNA Stability, RNA, Bacterial, RNA-Binding Proteins, Recombinant Fusion Proteins, Repressor Proteins

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          Abstract

          Small untranslated RNAs (sRNAs) perform a variety of important functions in bacteria. The 245 nucleotide sRNA of Escherichia coli, CsrC, was discovered using a genetic screen for factors that regulate glycogen biosynthesis. CsrC RNA binds multiple copies of CsrA, a protein that post-transcriptionally regulates central carbon flux, biofilm formation and motility in E. coli. CsrC antagonizes the regulatory effects of CsrA, presumably by sequestering this protein. The discovery of CsrC is intriguing, in that a similar sRNA, CsrB, performs essentially the same function. Both sRNAs possess similar imperfect repeat sequences (18 in CsrB, nine in CsrC), primarily localized in the loops of predicted hairpins, which may serve as CsrA binding elements. Transcription of csrC increases as the culture approaches the stationary phase of growth and is indirectly activated by CsrA via the response regulator UvrY. Because CsrB and CsrC antagonize CsrA activity and depend on CsrA for their synthesis, a csrB null mutation causes a modest compensatory increase in CsrC levels and vice versa. Homologues of csrC are apparent in several Enterobacteriaceae. The regulatory and evolutionary implications of these findings are discussed.

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          The global regulator CsrA of Escherichia coli is a specific mRNA-binding protein.

          The csrA gene encodes a global regulatory protein which facilitates glgC mRNA decay in vivo. A purified recombinant CsrA protein was found to inhibit in vitro glg (glycogen biosynthesis) gene expression posttranscriptionally and bind specifically to a glgC runoff transcript without causing its decay. Our results provide further insight into the mechanism by which CsrA functions as an mRNA decay factor.
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            The product of the pleiotropic Escherichia coli gene csrA modulates glycogen biosynthesis via effects on mRNA stability.

            The carbon storage regulator gene, csrA, modulates the expression of genes in the glycogen biosynthesis and gluconeogenesis pathways in Escherichia coli and has been cloned, mapped and sequenced (T. Romeo, M. Gong, M.Y. Liu, and A.M. Brun-Zinkernagel, J. Bacteriol. 175:4744-4755, 1993; T. Romeo and M. Gong, J. Bacteriol. 175:5740-5741, 1993). We have now conducted experiments that begin to elucidate a unique mechanism for csrA-mediated regulation. Steady-state levels of glgC transcripts, encoding ADP-glucose pyrophosphorylase, were elevated by up to sixfold in a csrA::kanR mutant and were less than 6.5% of wild-type levels in a strain containing pCSR10 (csrA+), as shown by S1 nuclease protection analysis. The rate of chemical decay of these transcripts after adding rifampin to cultures was dramatically reduced by the csrA::kanR mutation. Deletion studies of a glgC'-'lacZ translational fusion demonstrated that the region surrounding the initiation codon was important for csrA-mediated regulation and indicated that neither csrA-mediated regulation nor stationary phase induction of glgC expression originates at the level of transcript initiation. Cell-free (S-200) extracts containing the CsrA gene product potently and specifically inhibited the in vitro transcription-translation of glg genes. The deduced amino acid sequence of CsrA was found to contain the KH motif, which characterizes a subset of diverse RNA-binding proteins. The results indicate that CsrA accelerates net 5'-to-3' degradation of glg transcripts, potentially through selective RNA binding.
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              Coordinate genetic regulation of glycogen catabolism and biosynthesis in Escherichia coli via the CsrA gene product.

              The carbon storage regulator gene, csrA, encodes a factor which negatively modulates the expression of the glycogen biosynthetic gene glgC by enhancing the decay of its mRNA (M. Y. Liu, H. Yang, and T. Romeo, J. Bacteriol. 177:2663-2672, 1995). When endogenous glycogen levels in isogenic csrA+ and csrA::kanR strains were quantified during the growth curve, both the rate of glycogen accumulation during late exponential or early stationary phase and its subsequent rate of degradation were found to be greatly accelerated by the csrA::kanR mutation. The expression of the biosynthetic genes glgA (glycogen synthase) and glgS was observed to be negatively modulated via csrA. Thus, csrA is now known to control all of the known glycogen biosynthetic genes (glg), which are located in three different operons. Similarly, the expression of the degradative enzyme glycogen phosphorylase, which is encoded by glgY, was found to be negatively regulated via csrA in vivo. The in vitro transcription-translation of glgY was also specifically inhibited by the purified CsrA gene product. These results demonstrate that localization of glycogen biosynthetic and degradative genes within the Escherichia coli glgCAY operon facilitates their coordinate genetic regulation, as previously hypothesized (T. Romeo, A. Kumar, and J. Preiss, Gene 70:363-376, 1988). The csrA gene did not affect glycogen debranching enzyme, which is now shown to be encoded by the gene glgX.
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