5 years ago

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

Mura, C., K. A., Stanek
Hfq 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{middle dot}{middle dot}{middle dot}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{middle dot}{middle dot}{middle dot}RNA interactions (in vitro). Indeed, crystallographic and biophysical 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{middle dot}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.

Publisher URL: http://biorxiv.org/cgi/content/short/150672v1

DOI: 10.1101/150672

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