title: Conserved arginines on the rim of Hfq catalyze base pair formation and exchange.
abstract: The Sm-like protein Hfq is required for gene regulation by small RNAs (sRNAs) in bacteria and facilitates base pairing between sRNAs and their mRNA targets. The proximal and distal faces of the Hfq hexamer specifically bind sRNA and mRNA targets, but they do not explain how Hfq accelerates the formation and exchange of RNA base pairs. Here, we show that conserved arginines on the outer rim of the hexamer that are known to interact with sRNA bodies are required for Hfq's chaperone activity. Mutations in the arginine patch lower the ability of Hfq to act in sRNA regulation of rpoS translation and eliminate annealing of natural sRNAs or unstructured oligonucleotides, without preventing binding to either the proximal or distal face. Stopped-flow FRET and fluorescence anisotropy show that complementary RNAs transiently form a ternary complex with Hfq, but the RNAs are not released as a double helix in the absence of rim arginines. RNAs bound to either face of Hfq quench the fluorescence of a tryptophan adjacent to the arginine patch, demonstrating that the rim can simultaneously engage two RNA strands. We propose that the arginine patch overcomes entropic and electrostatic barriers to helix nucleation and constitutes the active site for Hfq's chaperone function.
authors.affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA and Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892-5430, USA.
authors.forename: Subrata
authors.initials: S
authors.lastname: Panja
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Schu
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Woodson
mesh_terms: D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D020134:Catalytic Domain; D017124:Conserved Sequence; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D035001:Host Factor 1 Protein / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D014176:Protein Biosynthesis; D059370:RNA Folding; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D012329:RNA, Bacterial; D012333:RNA, Messenger; D058727:RNA, Small Untranslated; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; D001120:Arginine; D008239:Lysine