E. coli DNA associated with isolated Hfq interacts with Hfq's distal surface and C-terminal domain.
E. coli DNA associated with isolated Hfq interacts with Hfq's distal surface and C-terminal domain.
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DOI：
10.1016/j.bbagrm.2010.06.007
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发表时间：
2010-08
影响因子：
4.7
通讯作者：
Wartell, Roger M.
中科院分区：
文献类型：
--
作者：
Updegrove, Taylor B.;Correia, John J.;Galletto, Roberto;Bujalowski, Wlodzimierz;Wartell, Roger M.
RNA结合蛋白Hfq在转录后水平上作为基因表达调节剂的功能已被广泛研究。虽然大多数Hfq研究集中在蛋白质与sRNA和mRNA的相互作用，但已经观察到Hfq与DNA的结合，但探索较少。在从大肠杆菌中分离Hfq的过程中，我们在多步纯化后发现了与该蛋白相关的基因组DNA片段。从与Hfq相关的DNA片段中扩增出41个片段，测定其序列。预测大部分DNA片段具有显著的螺旋轴曲率，并且来自与膜蛋白相关的基因，这是非特异性结合意想不到的特征。通过分析性超离心分析表明，rA 18与Hfq的结合破坏了Hfq-DNA相互作用。后一种观察结果表明Hfq与DNA的结合涉及其远端表面。凝胶迁移率变动分析表明，Hfq远端表面上的单个氨基酸突变抑制Hfq与双链体DNA的结合，而六聚体近端表面和外周上的七个突变中有六个没有阻止Hfq结合。两个C-末端结构域部分被去除的突变Hfq也不能与DNA结合。根据凝胶迁移率变动测定，野生型Hfq与几种双链体DNA结合的表观Kd估计为约400 nM。
The RNA-binding protein Hfq has been studied extensively for its function as a modulator of gene expression at the post-transcriptional level. While most Hfq studies have focused on the protein’s interaction with sRNAs and mRNAs, Hfq binding to DNA has been observed but is less explored. During the isolation of Hfq from Escherichia coli, we found genomic DNA fragments associated with the protein after multiple steps of purification. Sequences of 41 amplified segments from the DNA fragments associated with Hfq were determined. A large fraction of the DNA segments were predicted to have significant helical axis curvature and were from genes associated with membrane proteins, characteristics unexpected for non-specific binding. Analysis by analytical ultracentrifugation indicated that rA18 binding to Hfq disrupts Hfq-DNA interactions. The latter observation suggests Hfq binding to DNA involves its distal surface. This was supported by a gel mobility shift assay that showed single amino acid mutations on the distal surface of Hfq inhibited Hfq binding to duplex DNA, while six of seven mutations on the proximal surface and outer circumference of the hexamer did not prevent Hfq binding. Two mutated Hfq which have portions of their C-terminal domain removed also failed to bind to DNA. The apparent Kd for binding wild type Hfq to several duplex DNA was estimated from a gel mobility shift assay to be ~400 nM.
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