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首页> 外文期刊>Molecular and Cellular Biology >Variations in template protection by the RNA polymerase II transcription complex during the initiation process.
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Variations in template protection by the RNA polymerase II transcription complex during the initiation process.

机译：在起始过程中，RNA聚合酶II转录复合物对模板的保护作用有所不同。

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Preinitiation complexes (complex 0) or complexes which either made 2 or an average of 10 phosphodiester bonds (complexes 2 and 10, respectively) were assembled in vitro on the adenovirus 2 major late promoter. Each of the complexes was digested extensively with DNase I; the protected DNAs were purified and hybridized in a series of end-labeled oligonucleotides homologous to sequences on the coding or noncoding strands near the initiation site. The hybrids were then extended with reverse transcriptase to map the extent of template protection conferred by proteins in the complex. The downstream protection edge revealed by this approach was approximately +30, +25, and +35 for complexes 0, 2, and 10, respectively. We subsequently found that the apparent inward movement of the downstream protection boundary on initiation could be produced by satisfying the energy requirement for transcription initiation (i.e., by treating with ATP or dATP). The downstream boundary change occurred as rapidly as we could perform the test (less than 60 s) and was not blocked by alpha-amanitin. DNAs from trimmed complexes 0, 2, or 10 all supported extension to a single upstream edge at about position -42. Upstream protection was stable in the preinitiation complex, but when postinitiation complexes were incubated for extended periods, protection of the entire upstream region was lost. This decay of upstream protection, like the movement of the downstream boundary, was found to result from exposure to ATP or dATP. Unlike the downstream boundary movement, however, the upstream change was relatively slow; about 15 min was required to lose one-half of the protection.
机译：在腺病毒2主要晚期启动子上，体外组装预起始复合物（复合物0）或形成2个或平均10个磷酸二酯键的复合物（分别为复合物2和10）。每种复合物都用DNase I进行了广泛的消化。纯化被保护的DNA，并在一系列与起始位点附近的编码或非编码链上的序列同源的末端标记的寡核苷酸中杂交。然后用逆转录酶扩展杂合体以定位复合物中蛋白质赋予的模板保护程度。对于复合物0、2和10，该方法揭示的下游保护边缘分别约为+ 30，+ 25和+35。随后我们发现，通过满足转录起始的能量需求（即，通过用ATP或dATP处理），可以产生起始时下游保护边界的明显向内运动。下游边界变化发生的速度与我们进行测试的时间一样快（少于60 s），并且未被α-amanitin阻止。来自修剪的复合物0、2或10的DNA均支持延伸至约-42位的单个上游边缘。在启动前复合物中上游保护是稳定的，但是在启动后复合物长时间孵育后，整个上游区域的保护就失去了。发现上游保护作用的这种减弱，就像下游边界的运动一样，是由于暴露于ATP或dATP引起的。但是，与下游边界运动不同，上游变化相对较慢。大约需要15分钟才能失去一半的保护。

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