Safer B, Yang L, Tolunay H E, Anderson W F
Proc Natl Acad Sci U S A. 1985 May;82(9):2632-6. doi: 10.1073/pnas.82.9.2632.
Distinct RNA polymerase II transcription preinitiation, initiation, and elongation complexes can be formed in vitro on cloned adenovirus 2 DNA sequences containing the major late promoter. These transcription complexes are stable and can be rapidly isolated by gel filtration of HeLa whole cell extracts. In the absence of exogenous nucleotides and under appropriate salt conditions, a stable but transcriptionally incomplete preinitiation complex is formed. When this complex is incubated in the presence of adenosine or deoxyadenosine triphosphates, the beta-gamma phosphodiester bond is hydrolyzed, and RNA polymerase II joins the complex, thereby converting it into a stable initiation complex capable of forming (but prior to the formation of) the first phosphodiester bond. When this complex is isolated and incubated in the presence of all four nucleoside triphosphates, it is converted into an elongation complex that then permits the synthesis of phosphodiester bonds and the correct run-off transcript. A limiting transcription component is sequestered in the preinitiation complex. This factor is released upon elongation and can reassociate with new DNA templates during subsequent rounds of initiation. Therefore, class II genes do not appear to form activated transcription units stable for multiple rounds of transcription; rather, their transcriptional activity may be controlled in part by regulating the association of transcription factors at each initiation event.
在含有主要晚期启动子的克隆腺病毒2 DNA序列上，可在体外形成不同的RNA聚合酶II转录起始前、起始和延伸复合物。这些转录复合物是稳定的，可通过对HeLa全细胞提取物进行凝胶过滤快速分离。在没有外源核苷酸且处于适当盐条件下时，会形成稳定但转录不完全的起始前复合物。当该复合物在腺苷或脱氧腺苷三磷酸存在下孵育时，β-γ磷酸二酯键被水解，RNA聚合酶II加入该复合物，从而将其转化为能够形成（但在形成之前）第一个磷酸二酯键的稳定起始复合物。当分离该复合物并在所有四种核苷三磷酸存在下孵育时，它会转化为延伸复合物，进而允许磷酸二酯键的合成和正确的连续转录物。一个有限的转录成分被隔离在起始前复合物中。该因子在延伸时释放，并可在随后的起始轮次中与新的DNA模板重新结合。因此，II类基因似乎不会形成对多轮转录稳定的活化转录单元；相反，它们的转录活性可能部分通过在每个起始事件中调节转录因子的结合来控制。
