Despite Similar Binding to the Hfq Protein Regulatory RNAs Widely Differ in Their Competition Performance
Despite Similar Binding to the Hfq Protein Regulatory RNAs Widely Differ in Their Competition Performance
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DOI：
10.1021/bi102043f
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发表时间：
2011-05-31
期刊：
影响因子：
2.9
通讯作者：
Olejniczak, Mikolaj
中科院分区：
文献类型：
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作者：
Olejniczak, Mikolaj
使用高通量双滤器保留测定比较了九种非编码调节 RNA (sRNA) 与大肠杆菌 Hfq 蛋白的结合。尽管这些 sRNA 具有不同的长度、序列和二级结构，但它们的 Hfq 结合亲和力却惊人地一致。对与 Hfq 突变体结合的 sRNA 的分析表明，Hfq 的近端面（称为 DsrA RNA 的结合位点）是通用的 sRNA 结合位点。此外，所有 sRNA 与 Hfq 的结合率相似，仅受扩散速率的限制，而在稀释实验中测得的解离速率则一致缓慢。尽管如此，数据显示，sRNA 在竞争 Hfq 的表现以及促进其他 sRNA 从 Hfq 解离的能力方面存在层次结构。 sRNA 与该蛋白质结合的盐依赖性也有所不同。总的来说，结果表明，尽管不同的 sRNA 统一结合到 Hfq 上的同一位点，但它们在该蛋白质上的交换取决于竞争 sRNA 的身份。
The binding of nine noncoding regulatory RNAs (sRNAs) to the E. coli Hfq protein was compared using a high-throughput double filter retention assay. Despite the fact that these sRNAs have different lengths, sequences and secondary structures their Hfq binding affinities were surprisingly uniform. The analysis of sRNAs binding to Hfq mutants showed that the proximal face of Hfq, known as the binding site for DsrA RNA, is a universal sRNA binding site. Moreover, all sRNAs bound Hfq with similar association rates limited only by the rate of diffusion, while the rates of dissociation, measured in the dilution experiments, were uniformly slow. Despite that, the data showed that there was a hierarchy of sRNAs in regard to their performance in competition for access to Hfq and in their ability to facilitate the dissociation of other sRNAs from Hfq. The sRNAs also differed in their salt dependence of binding to this protein. Overall, the results suggest that despite the uniform binding of different sRNAs to the same site on Hfq their exchange on this protein is dependent on the identities of the competing sRNAs.