title: CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage.
abstract: Csr is a conserved global regulatory system that controls expression of several hundred Escherichia coli genes. CsrA protein represses translation of numerous genes by binding to mRNA and inhibiting ribosome access. CsrA also activates gene expression, although an activation mechanism has not been reported. CsrA activates flhDC expression, encoding the master regulator of flagellum biosynthesis and chemotaxis, by stabilizing the mRNA. Computer modelling, gel mobility shift and footprint analyses identified two CsrA binding sites extending from positions 1-12 (BS1) and 44-55 (BS2) of the 198 nt flhDC leader transcript. flhD'-'lacZ expression was reduced by mutations in csrA and/or the CsrA binding sites. The position of BS1 suggested that bound CsrA might inhibit 5' end-dependent RNase E cleavage of flhDC mRNA. Consistent with this hypothesis, CsrA protected flhDC leader RNA from RNase E cleavage in vitro and protection depended on BS1 and BS2. Primer extension studies identified flhDC decay intermediates in vivo that correspond to in vitro RNase E cleavage sites. Deletion of these RNase E cleavage sites resulted in increased flhD'-'lacZ expression. Data from mRNA decay studies and quantitative primer extension assays support a model in which bound CsrA activates flhDC expression by inhibiting the 5' end-dependent RNase E cleavage pathway.
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Yakhnin
authors.forename: Carol S
authors.initials: CS
authors.lastname: Baker
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Vakulskas
authors.forename: Helen
authors.initials: H
authors.lastname: Yakhnin
authors.forename: Igor
authors.initials: I
authors.lastname: Berezin
authors.forename: Tony
authors.initials: T
authors.lastname: Romeo
authors.forename: Paul
authors.initials: P
authors.lastname: Babitzke
mesh_terms: D020121:5' Untranslated Regions; D001483:Base Sequence; D001665:Binding Sites; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009876:Operon; D011485:Protein Binding; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; C093587:CsrA protein, E coli; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; C090552:flhC protein, E coli; C090553:flhD protein, E coli; D004722:Endoribonucleases; C022537:ribonuclease E