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

  • Kimberly A. Stanek
  • Cameron Mura
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1737)

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.

Keywords

Hfq Sm sRNA RNA chaperone RNA-binding protein Crystallization In vitro transcription 

Abbreviations

3D

Three-dimensional

AU

Asymmetric unit

CV

Column volume

DEPC

Diethyl pyrocarbonate

HDV

Hepatitis δ virus

HDVD

Hanging-drop vapor diffusion

IMAC

Immobilized metal affinity chromatography

MW

Molecular weight

MWCO

Molecular weight cut-off

nt

Nucleotide

PDB

Protein Data Bank

RNP

Ribonucleoprotein

RT

Room temperature

SDVD

Sitting-drop vapor diffusion

Notes

Acknowledgments

We thank L. Columbus (UVa) for helpful discussions. This work was funded by NSF Career award MCB–1350957.

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Authors and Affiliations

  1. 1.Department of ChemistryUniversity of VirginiaCharlottesvilleUSA

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