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

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


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.


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





Asymmetric unit


Column volume


Diethyl pyrocarbonate


Hepatitis δ virus


Hanging-drop vapor diffusion


Immobilized metal affinity chromatography


Molecular weight


Molecular weight cut-off




Protein Data Bank




Room temperature


Sitting-drop vapor diffusion



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


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© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of VirginiaCharlottesvilleUSA

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