Separation and partial characterization of three functional steps in transcription initiation by human RNA polymerase II.
Separation and partial characterization of three functional steps in transcription initiation by human RNA polymerase II.
复制标题
DOI：
10.1016/s0021-9258(17)39577-7
复制
发表时间：
1985-07
期刊：
影响因子：
--
通讯作者：
D. K. Hawley;R. Roeder
中科院分区：
文献类型：
--
作者：
D. K. Hawley;R. Roeder
我们已经使用Sarkosyl研究HeLa RNA聚合酶II在体外特异性转录起始的事件。基于对肌氨酰浓度的不同敏感性，我们定义了腺病毒主要晚期启动子启动的三个功能步骤：1。在0.015%肌氨酰存在下发生的模板定型步骤; 2.所述定向复合物向能够起始RNA链的“快速起始复合物”的核苷酸非依赖性转化，该步骤被0.015%的Sarkosyl阻断;和3.需要核苷三磷酸的步骤，将快速起始复合物转化为稳定起始复合物，并且对大于0.05%的肌氨酸浓度敏感。起始RNA链的随后延伸对Sarkosyl具有抗性，除了Sarkosyl在主要晚期帽位点下游约186个核苷酸的特定位点处引起暂停或过早终止。使用该测定法，我们进一步表征了这些步骤和所得的中间体复合物。
We have used Sarkosyl to study the events comprising specific transcription initiation in vitro by HeLa RNA polymerase II. On the basis of different sensitivities to the Sarkosyl concentration, we have defined three functional steps in initiation at the adenovirus major late promoter: 1. a template commitment step that occurs in the presence of 0.015% Sarkosyl; 2. a nucleotide-independent conversion of the committed complex to a “rapid start complex” capable of initiating an RNA chain, a step blocked by 0.015% Sarkosyl; and 3. a step that requires nucleoside triphosphates, converts the rapid start complex to a stably initiated complex, and is sensitive to Sarkosyl concentrations greater than 0.05%. The subsequent elongation of the initiated RNA chain is resistant to Sarkosyl, except that Sarkosyl causes pausing or premature termination at a specific site about 186 nucleotides downstream of the major late cap site. Using this assay, we have further characterized these steps and the resulting intermediate complexes.