Chapter title |
Producing Hfq/Sm Proteins and sRNAs for Structural and Biophysical Studies of Ribonucleoprotein Assembly
|
---|---|
Chapter number | 16 |
Book title |
Bacterial Regulatory RNA
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7634-8_16 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7633-1, 978-1-4939-7634-8
|
Authors |
Kimberly A. Stanek, Cameron Mura, Stanek, Kimberly A., Mura, Cameron |
Abstract |
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∙∙∙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 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·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. |
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