title: CsrA regulates translation of the Escherichia coli carbon starvation gene, cstA, by blocking ribosome access to the cstA transcript.
abstract: CsrA is a global regulator that binds to two sites in the glgCAP leader transcript, thereby blocking ribosome access to the glgC Shine-Dalgarno sequence. The upstream CsrA binding site (GCACACGGAU) was used to search the Escherichia coli genomic sequence for other genes that might be regulated by CsrA. cstA contained an exact match that overlapped its Shine-Dalgarno sequence. cstA was previously shown to be induced by carbon starvation and to encode a peptide transporter. Expression of a cstA'-'lacZ translational fusion in wild-type and csrA mutant strains was examined. Expression levels in the csrA mutant were approximately twofold higher when cells were grown in Luria broth (LB) and 5- to 10-fold higher when LB was supplemented with glucose. It was previously shown that cstA is regulated by the cyclic AMP (cAMP)-cAMP receptor protein complex and transcribed by Esigma(70). We investigated the influence of sigma(S) on cstA expression and found that a sigma(S) deficiency resulted in a threefold increase in cstA expression in wild-type and csrA mutant strains; however, CsrA-dependent regulation was retained. The mechanism of CsrA-mediated cstA regulation was also examined in vitro. Cross-linking studies demonstrated that CsrA is a homodimer. Gel mobility shift results showed that CsrA binds specifically to cstA RNA, while coupled-transcription-translation and toeprint studies demonstrated that CsrA regulates CstA synthesis by inhibiting ribosome binding to cstA transcripts. RNA footprint and boundary analyses revealed three or four CsrA binding sites, one of which overlaps the cstA Shine-Dalgarno sequence, as predicted. These results establish that CsrA regulates translation of cstA by sterically interfering with ribosome binding.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Dubey
authors.forename: Carol S
authors.initials: CS
authors.lastname: Baker
authors.forename: Kazushi
authors.initials: K
authors.lastname: Suzuki
authors.forename: A Daniel
authors.initials: AD
authors.lastname: Jones
authors.forename: Pallavi
authors.initials: P
authors.lastname: Pandit
authors.forename: Tony
authors.initials: T
authors.lastname: Romeo
authors.forename: Paul
authors.initials: P
authors.lastname: Babitzke
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002244:Carbon / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: C093587:CsrA protein, E coli; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; C121562:cstA protein, E coli; D002244:Carbon