Positional effects of AAN motifs in rpoS regulation by sRNAs and Hfq

Yi Peng, Toby J. Soper, Sarah A. Woodson

Research output: Contribution to journalArticle

Abstract

The Escherichia coli stationary phase transcription factor RpoS is translated in response to small noncoding RNAs (sRNAs), which base pair with the rpoS mRNA leader. The bacterial Sm-like protein Hfq anneals sRNAs with their mRNA targets by simultaneously binding the mRNA and sRNA. Intriguingly, Hfq is recruited to the rpoS leader via AAN motifs far upstream of the sRNA. SHAPE (selective 2′-hydroxyl acylation and primer extension) chemical footprinting showed that the rpoS leader is divided into a far upstream domain, an Hfq binding domain, and a downstream inhibitory stem-loop containing the sRNA and ribosome binding sites. To investigate how Hfq promotes sRNA-mRNA base pairing from a distance, we deleted the natural AAN Hfq binding site, and we inserted artificial AAN binding sites at various positions in the rpoS leader. All the relocated AAN motifs restored tight Hfq binding in vitro, but only insertion at the natural position restored Hfq-dependent sRNA annealing in vitro and sRNA regulation of rpoS translation in vivo. Furthermore, U-rich motifs in the downstream inhibitory domain stabilized the rpoS mRNA-Hfq complex and contributed to regulation of rpoS expression. We propose that the natural Hfq binding domain is optimal for positive regulation because it recruits Hfq to the mRNA and allows it to act on incoming sRNAs without opening the inhibitory stem-loop when sRNA is absent.

Original languageEnglish (US)
Pages (from-to)275-285
Number of pages11
JournalJournal of molecular biology
Volume426
Issue number2
DOIs
StatePublished - Jan 23 2014

Keywords

  • 5′ UTR
  • RNA chaperone
  • RNA-protein interactions
  • bacterial stress response
  • translational control

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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