Levin J R, Blake J J, Ganunis R A, Tullius T D
Departments of Biological Sciences and Chemistry, Goucher College, Baltimore, Maryland 21204, USA.
J Biol Chem. 2000 Mar 10;275(10):6885-93. doi: 10.1074/jbc.275.10.6885.
We have examined the effects of removing individual template nucleosides on promoter escape by Escherichia coli RNA polymerase in vitro. The ability of DNA templates containing random single nucleoside gaps generated by hydroxyl radical treatment to support the production of stable ternary transcription complexes was analyzed. On two templates containing different promoter and initial transcribed regions, we found that removal of nucleosides on the template strand in the region from -13 to at least +8 relative to the transcription start site interfered with ternary complex formation. The downstream border of this region varied for the two templates, suggesting an effect of the specific nucleotide sequence on the stability of intermediates in the promoter escape process. On the nontemplate strand, removal of nucleosides in the vicinity of the -10 consensus promoter element interfered with escape, whereas removal of nucleosides in the vicinity of the transcription start site actually enhanced the yield of ternary complexes. On one template, removal of nucleosides in an A-tract containing region upstream of the promoter caused a significant decrease in promoter escape, consistent with previous suggestions that contacts between this region and the RNA polymerase play a role in promoter binding and/or initiation.
我们已经在体外研究了去除单个模板核苷对大肠杆菌RNA聚合酶启动子逃逸的影响。分析了经羟基自由基处理产生随机单核苷间隙的DNA模板支持稳定三元转录复合物产生的能力。在两个包含不同启动子和初始转录区域的模板上，我们发现相对于转录起始位点，在-13至至少+8区域的模板链上去除核苷会干扰三元复合物的形成。这两个模板的该区域下游边界有所不同，表明特定核苷酸序列对启动子逃逸过程中中间体稳定性有影响。在非模板链上，去除-10共有启动子元件附近的核苷会干扰逃逸，而去除转录起始位点附近的核苷实际上会提高三元复合物的产量。在一个模板上，去除启动子上游富含A区域中的核苷会导致启动子逃逸显著降低，这与之前认为该区域与RNA聚合酶之间的接触在启动子结合和/或起始中起作用的观点一致。
