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Bacterial Regulatory RNA pp 273–299Cite as

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

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Part of the book series: Methods in Molecular Biology ((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.

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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

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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. Department of Chemistry, University of Virginia, Charlottesville, VA, USA

    Kimberly A. Stanek & Cameron Mura

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Correspondence to Kimberly A. Stanek or Cameron Mura .

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  1. Université Paris Diderot & LLB/CEA/CNRS, Gif-sur-Yvette, France

    Véronique Arluison

  2. Laboratorio de Bioquímica, Microbiología e Interacciones, Biológicas en el Suelo, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes-CONICET, Bernal, Argentina

    Claudio Valverde

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Stanek, K.A., Mura, C. (2018). Producing Hfq/Sm Proteins and sRNAs for Structural and Biophysical Studies of Ribonucleoprotein Assembly. In: Arluison, V., Valverde, C. (eds) Bacterial Regulatory RNA. Methods in Molecular Biology, vol 1737. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7634-8_16

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  • DOI: https://doi.org/10.1007/978-1-4939-7634-8_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7633-1

  • Online ISBN: 978-1-4939-7634-8

  • eBook Packages: Springer Protocols

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