Stanek Kimberly A, Mura Cameron
Department of Chemistry, University of Virginia, Charlottesville, VA, USA.
Methods Mol Biol. 2018;1737:273-299. doi: 10.1007/978-1-4939-7634-8_16.
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是一种细菌RNA结合蛋白，在基因表达的转录后调控中发挥关键作用。与其他Sm蛋白一样，Hfq组装成环形盘状结构，以不同的亲和力和序列特异性程度结合RNA。通过同时结合调控性小RNA（sRNA）和mRNA靶标，Hfq六聚体促进了高效的RNA∙∙∙RNA相互作用；这种类似伴侣蛋白的功能的通用性使Hfq成为许多基于sRNA的调控网络的核心。Hfq在多种细胞途径中至关重要，包括应激反应、群体感应和生物膜形成，这激发了对其功能和生理学（体内）的遗传学和“RNA组学”研究，以及对Hfq∙∙∙RNA相互作用（体外）的生化和结构分析。事实上，晶体学和生物物理研究首先将Hfq确立为系统发育上保守的Sm超家族的成员。晶体学和其他生物物理方法能够在原子水平上详细阐明Hfq的RNA结合特性，但这些方法对样品有严格的要求，即：重建和表征Hfq·RNA复合物需要大量性能良好（足够纯度、均一性）的Hfq和RNA样品（sRNA、mRNA片段、短寡核糖核苷酸，甚至单核苷酸）。本章涵盖了此类材料的制备，特别关注用于结晶实验的重组Hfq蛋白。
