CsrA activates flhDC expression by protecting flhDC mRNA from RNase E‐mediated cleavage

Abstract

Summary 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 modeling, 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.

Regulation of Bacterial Virulence by Csr (Rsm) Systems

“…Yakhnin et al later identified the molecular mechanism of CsrA-mediated activation; CsrA binds to two sites within the flhDC RNA leader, which prevents 5= end-dependent cleavage of this transcript by RNase E (Fig. 2) (47). While effects on translation often cause secondary and ␣-helices depicted as arrows and cylinders, respectively, and amino acid sequence comparisons are shown immediately below.…”

“…(F) E. coli CsrA stabilizes flhDC by preventing endonuclease cleavage by RNase E. (Top) CsrA binds to sites (GGA) present in the single-stranded region of two RNA hairpins located at the 5= end of flhDC (47). CsrA binding prevents 5= end-dependent cleavage by RNase E. (Bottom) In the absence of CsrA, RNase E binds to the 5= monophosphorylated end of flhDC and performs several cleavages that initiate turnover of the transcript (47).…”

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli

“…CsrA regulates the expression of genes involved in lifestyle transitions. In Escherichia coli, CsrA activates glycolysis and central carbon pathways (7)(8)(9)(10)(11)(12)(13) and motility (14,15). Conversely, it represses gluconeogenesis (7), glycogen biosynthesis (16)(17)(18)(19)(20), biofilm formation (21)(22)(23)(24), the stringent response (25), and expression of genes involved in other stress resistance and stationary-phase processes, e.g., cstA, hfq, cel, sdiA, and nhaR (24,(26)(27)(28)(29)(30).…”

Dual function of the McaS small RNA in controlling biofilm formation

“…These enzymes have been shown to regulate motility and biofilm formation as well as PgaD expression at the posttranscriptional level by altering cyclic di-GMP (c-di-GMP) levels (Jonas et al 2008;Boehm et al 2009;Steiner et al 2013). CsrA also binds the flhD 59 UTR and activates expression of this transcription regulator through protection from cleavage by RNase E (Yakhnin et al 2013). Additional CsrA-mediated effects on biofilm formation might be due to observed CsrA repression of Hfq expression (Baker et al 2007), which could alter the stabilities of sRNA regulators of biofilm formation.…”