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Abstract:
The homohexameric (L)Sm protein Hfq is a central mediator of small RNA-based gene regulation in bacteria. Hfq recognizes small regulatory RNAs (sRNAs) specifically, despite their structural diversity. This specificity could not be explained by previously described RNA-binding modes of Hfq. Here we present a distinct and preferred mode of Hfq-RNA interaction that involves the direct recognition of a uridine-rich RNA 3' end. This feature is common in bacterial RNA transcripts as a consequence of Rho-independent transcription termination and hence likely contributes significantly to the general recognition of sRNAs by Hfq. Isothermal titration calorimetry shows nanomolar affinity between Salmonella typhimurium Hfq and a hexauridine substrate. We determined a crystal structure of the complex that reveals a constricted RNA backbone conformation in the proximal RNA-binding site of Hfq, allowing for a direct protein contact of the 3' hydroxyl group. A free 3' hydroxyl group is crucial for the high-affinity interaction with Hfq also in the context of a full-length sRNA substrate, RybB. The capacity of Hfq to occupy and sequester the RNA 3' end has important implications for the mechanisms by which Hfq is thought to affect sRNA stability, turnover, and regulation.
