Positioning of the start site in the initiation of transcription by bacteriophage T7 RNA polymerase
Positioning of the start site in the initiation of transcription by bacteriophage T7 RNA polymerase
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DOI：
10.1006/jmbi.1997.1199
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发表时间：
1997-09-12
影响因子：
5.6
通讯作者：
Martin, CT
中科院分区：
文献类型：
--
作者：
Weston, BF;Kuzmine, I;Martin, CT
各种聚合酶结构的测定激发了人们对了解多核苷酸模板如何与活性位点相互作用的兴趣。在不依赖引物的转录起始中，出现了一个额外的问题，即复合物如何将模板的前两个碱基独特地引导到活性位点。最近对来自噬菌体 T7 的模型 RNA 聚合酶的研究表明，上游双链体接触至少提供了一些结合特异性，并表明该酶在转录起始位点附近的熔化环境中与模板链相互作用。目前的工作探讨了模板链在起始过程中前两个模板碱基定位中的作用。结果表明，这种定位不会限制稳态周转，并且在起始位点上游三或四个碱基处插入非常大且灵活的接头对起始位点选择的保真度没有显着影响。然而，紧邻起始位点插入接头确实会显着降低起始位点选择的保真度（如位置 +2 处错误起始的大量增加所证明的，而观察到的正确起始率几乎没有变化），这表明一些非转录模板 DNA 确实有助于将前两个模板碱基定位到 RNA 聚合酶的活性位点中。最后，在位置-1处掺入脱碱基位点会导致起始保真度出现类似的降低，这表明在位置-1和+1处碱基堆叠的作用。 (C) 1997 学术出版社有限公司。
The determination of various polymerase structures has sparked interest in understanding how the polynucleotide template interacts with the active site. In the primer-independent initiation of transcription, an additional question arises as to how the complex directs the first two bases of the template uniquely into the active site. Recent studies in the model RNA polymerase from bacteriophage T7 demonstrate that upstream duplex contacts provide at least some of the binding specificity and suggest that the enzyme interacts with the template strand in a melted context near the start site for transcription. The current work probes the role of the template strand in positioning of the first two templating bases during initiation. The results suggest that such positioning is not rate-limiting in steady-state turnover, and that the insertion of a very large and flexible linker three or four bases upstream of the start site has no significant effect on the fidelity of start site selection. The insertion of linkers immediately adjacent to the start site, however, does significantly decrease the fidelity of start site selection (as evidenced by a large increase in misinitiation at position +2, with little change in the observed rate of correct initiation), suggesting that some of the non-transcribed template DNA does help to position the first two templating bases into the active site of the RNA polymerase. Finally, incorporation of an abasic site at position -1 yields a similar decrease in initiation fidelity, suggesting a role for stacking of the bases at positions -1 and +1. (C) 1997 Academic Press Limited.