Producing Hfq/Sm Proteins and sRNAs for Structural and Biophysical Studies of Ribonucleoprotein Assembly

Abstract

AbstractHfq is a bacterial RNA-binding protein that plays key roles in the post–transcriptional regulation of gene expression. Like other Sm proteins, Hfq assembles into toroidal discs that bind RNAs with varying affinities and degrees of sequence specificity. By simultaneously binding to a regulatory small RNA (sRNA) and an mRNA target, Hfq hexamers facilitate productive RNA⋯RNA interactions; the generic nature of this chaperone-like functionality makes Hfq a hub in many sRNA-based regulatory networks. That Hfq is crucial in diverse cellular pathways—including stress response, quorum sensing and biofilm formation— has motivated genetic and ‘RNAomic’ studies of its function and physiology (in vivo), as well as biochemical and structural analyses of Hfq⋯RNA interactions (in vitro). Indeed, crystallographic and bio-physical studies first established Hfq as a member of the phylogenetically-conserved Sm superfamily. Crystallography and other biophysical methodologies enable the RNA-binding properties of Hfq to be elucidated in atomic detail, but such approaches have stringent sample requirements, viz.: reconstituting and characterizing an Hfq•RNA complex requires ample quantities of well-behaved (sufficient purity, homogeneity) specimens of Hfq and RNA (sRNA, mRNA fragments, short oligoribonucleotides, or even single nucleotides). The production of such materials is covered in this Chapter, with a particular focus on recombinant Hfq proteins for crystallization experiments.Abbreviations3Dthree-dimensionalAUasymmetric unitCVcolumn volumeDEPCdiethyl pyrocarbonateHDVhepatitis δ virusHDVDhanging-drop vapor diffusionIMACimmobilized metal affinity chromatographyMWmolecular weightMWCOmolecular weight cut-offntnucleotidePDBProtein Data BankRNPribonucleoproteinRTroom temperatureSDVDsitting-drop vapor diffusionJournal formatMethods in Molecular Biology (Springer Protocols series); this volume is entitled “Bacterial Regulatory RNA: Methods and Protocols”; an author guide is linked at http://www.springer.com/series/7651