Structural Models of Bacterial RNA Chaperone Hfq in Complex with the RNA Targets

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Date
2014-11-21
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Johns Hopkins University
Abstract
Bacterial Sm protein Hfq is required for translation regulation by small non- coding RNAs (sRNAs), which play a key role in bacterial environmental adaptation and pathogenic pathways. Hfq promotes anti-sense base pairing of the sRNAs that inhibits or activates translation of the mRNA target. For the positive regulation of stress response regulator rpoS, Hfq distal surface binds to an upstream (AAN) motif in the mRNA leader, while the proximal surface and lateral rim bind to the U-rich sequences in the sRNAs. I found that the Hfq activity requires the (AAN) motif located < 80 nt upstream of the sRNA annealing site in the rpoS leader, because Hfq lateral rim directly binds a downstream U5 motif, as shown by SHAPE, in vivo LacZ activity assays, and in vitro binding assays. Small angle X ray scattering (SAXS) showed that Hfq folds the rpoS mRNA leader into a compact tertiary conformation. Finally, I generated all-atom structural models of rpoS•Hfq complex by combining MC-Sym structure prediction, rigid body modeling, and Monte Carlo simulation based on the SAXS data. The dynamic interaction models suggest that Hfq binds the U5 motif to position and unwind the inhibitory stem for sRNA entry and annealing, and that Hfq disengages from the downstream domain to release the RNA duplex for translation initiation. I also discovered that Hfq does not bind a U-rich motif in the flhD leader or restructures the RNA significantly, causing only moderate enhancement for sRNA annealing efficiency. Moreover, Hfq binds to various sRNAs by distinctive modes depending on the presence of the A-rich or U-rich sequences; however, annealing activity relies more on the complementarity between the base pairing RNAs than the binding mode for a specific RNA.
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Lsm protein, RNA-protein interactions, SAXS, RNase foot printing, SHAPE, bacterial post transcriptional control, small noncoding RNA
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