title: CsrA inhibits translation initiation of Escherichia coli hfq by binding to a single site overlapping the Shine-Dalgarno sequence.
abstract: Csr (carbon storage regulation) of Escherichia coli is a global regulatory system that consists of CsrA, a homodimeric RNA binding protein, two noncoding small RNAs (sRNAs; CsrB and CsrC) that function as CsrA antagonists by sequestering this protein, and CsrD, a specificity factor that targets CsrB and CsrC for degradation by RNase E. CsrA inhibits translation initiation of glgC, cstA, and pgaA by binding to their leader transcripts and preventing ribosome binding. Translation inhibition is thought to contribute to the observed mRNA destabilization. Each of the previously known target transcripts contains multiple CsrA binding sites. A position-specific weight matrix search program was developed using known CsrA binding sites in mRNA. This search tool identified a potential CsrA binding site that overlaps the Shine-Dalgarno sequence of hfq, a gene that encodes an RNA chaperone that mediates sRNA-mRNA interactions. This putative CsrA binding site matched the SELEX-derived binding site consensus sequence in 8 out of 12 positions. Results from gel mobility shift and footprint assays demonstrated that CsrA binds specifically to this site in the hfq leader transcript. Toeprint and cell-free translation results indicated that bound CsrA inhibits Hfq synthesis by competitively blocking ribosome binding. Disruption of csrA caused elevated expression of an hfq'-'lacZ translational fusion, while overexpression of csrA inhibited expression of this fusion. We also found that hfq mRNA is stabilized upon entry into stationary-phase growth by a CsrA-independent mechanism. The interaction of CsrA with hfq mRNA is the first example of a CsrA-regulated gene that contains only one CsrA binding site.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Carol S
authors.initials: CS
authors.lastname: Baker
authors.forename: Lél A
authors.initials: LA
authors.lastname: Eöry
authors.forename: Helen
authors.initials: H
authors.lastname: Yakhnin
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Mercante
authors.forename: Tony
authors.initials: T
authors.lastname: Romeo
authors.forename: Paul
authors.initials: P
authors.lastname: Babitzke
mesh_terms: D019677:Artificial Gene Fusion; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D035001:Host Factor 1 Protein / Q000235:genetics*; D016254:Mutagenesis, Insertional; D010442:Peptide Chain Initiation, Translational; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
chemical_list: C093587:CsrA protein, E coli; D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D001616:beta-Galactosidase