Mapping the spatial neighborhood of the regulatory 6S RNA bound to Escherichia coli RNA polymerase holoenzyme.
Mapping the spatial neighborhood of the regulatory 6S RNA bound to Escherichia coli RNA polymerase holoenzyme.
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绘制与大肠杆菌 RNA 聚合酶全酶结合的调节 6S RNA 的空间邻域
DOI：
10.1016/j.jmb.2013.07.008
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发表时间：
2013
影响因子：
5.6
通讯作者：
Wagner R
中科院分区：
文献类型：
--
作者：
Steuten B;Setny P;Zacharias M;Wagner R
细菌 6S RNA 与充当转录调节因子的 RNA 聚合酶特异性相互作用。到目前为止，尚未对 RNA 聚合酶三维结构中非编码 RNA 的空间排列进行详细表征。在这里，我们展示了用化学核酸酶 FeBABE 束缚在 RNA 聚合酶 σ70 亚基的不同位置上获得的结果。 6S RNA 复合物是由一组 RNA 聚合酶形成的，其中切割试剂已与靠近参与启动子识别的区域的 σ70 单半胱氨酸变体融合。鉴定了 6S RNA 结构内 FeBABE 诱导的切割位点，表明 σ70 单半胱氨酸侧链与 6S RNA 结构的确定位置之间存在紧密的空间邻域。我们的分析表明 6S RNA 内部发夹和 σ70 结构域 4.2 之间非常接近，通常参与 − 35 启动子 DNA 的识别。中央气泡两侧的内部 6S RNA 茎结构的确定部分位于保守的 σ70 结构域 3.1、2.3 和 2.1 附近，这些结构域与 − 10 和 − 35 元件之间的 DNA 启动子的结合和熔解有关。此外，我们发现6S RNA的U44位于RNA聚合酶活性位点（σ70结构域3.2）附近，与其在RNA指导的pRNA转录中作为起始核苷酸的功能完全一致。在 6S RNA 和 σ70 区域 1.2 之间或 σ70 和 6S RNA 闭合茎结构（残基 1-41 和 144-184）之间未检测到相邻接触。结果用于将6S RNA的结构模型与已知的大肠杆菌σ70RNA聚合酶全酶的三维结构对接。
Bacterial 6S RNA interacts specifically with RNA polymerase acting as transcriptional regulator. Until now, no detailed characterization of the spatial arrangement of the non-coding RNA within the three-dimensional structure of RNA polymerase has been performed. Here we present results obtained with the chemical nuclease FeBABE tethered to distinct positions of RNA polymerase σ70subunit. 6S RNA complexes were formed with a collection of RNA polymerases, where the cleavage reagent had been fused to σ70single-cysteine variants close to regions involved in promoter recognition. FeBABE-induced cleavage sites within the 6S RNA structure were identified, indicating close spatial neighborhood between σ70single-cysteine side chains and defined positions of the 6S RNA structure. Our analysis demonstrates close proximity between the 6S RNA internal hairpin and σ70domain 4.2, normally involved in recognition of − 35 promoter DNA. Defined sections of the internal 6S RNA stem structure flanking the central bubble are positioned near conserved σ70domains 3.1, 2.3 and 2.1, which are implicated in binding and melting DNA promoters between the − 10 and − 35 elements. Moreover, we show that U44 of 6S RNA is located near RNA polymerase active site (σ70domain 3.2), fully consistent with its function as starting nucleotide in RNA-directed pRNA transcription. No neighboring contacts were detected between 6S RNA and σ70region 1.2 or between σ70and the 6S RNA closing stem structure (residues 1–41 and 144–184). Results were used to dock a structural model of 6S RNA to the known three-dimensional structure ofEscherichia coliσ70RNA polymerase holoenzyme.
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