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J Mol Biol. 2014 Jan 23;426(2):275-85. doi: 10.1016/j.jmb.2013.08.026. Epub 2013 Sep 16.

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

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Program in Cellular, Molecular and Developmental Biology and Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA. Electronic address:


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.


5′ UTR; EDTA; RNA chaperone; RNA–protein interactions; bacterial stress response; ethylenediaminetetraacetic acid; sRNA; small RNA; translational control

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