Studies of in vitro transcription by calf thymus RNA polymerase II using a novel duplex DNA template.
Studies of in vitro transcription by calf thymus RNA polymerase II using a novel duplex DNA template.
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使用新型双链 DNA 模板研究小牛胸腺 RNA 聚合酶 II 的体外转录。
DOI：
10.1016/s0021-9258(18)34669-6
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
M. Chamberlin
中科院分区：
文献类型：
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作者：
T. Kadesch;M. Chamberlin
在T7噬菌体DNA的3 '-OH末端添加10至100个寡脱氧胞苷酸残基，可产生一种高效的模板，用于用纯化的小牛胸腺RNA聚合酶II进行体外转录。转录在这样的模板的寡聚体（dC）末端快速且选择性地启动，并且基本上所有的活性RNA聚合酶II分子随后致力于长时间的RNA链延伸。这允许直接研究RNA链延伸和终止反应，并且还允许测定存在的活性RNA聚合酶II的浓度。在我们目前的制备中，15%至25%的RNA聚合酶分子在这些反应中具有活性。即使在饱和底物浓度下，小牛胸腺RNA聚合酶II的RNA链延伸也相对缓慢（7个核苷酸/s）。体外延伸过程似乎是不连续的，沿着DNA沿着的聚合酶分子在某些序列处停顿了相当长的时间。在某些位点，RNA链终止的频率很低，但可以测量;然而，大多数延伸转录本可以生长到很大的尺寸（超过6000个核苷酸）。令人惊讶的是，超过60%的活性小牛胸腺RNA聚合酶II分子在体外转录过程中形成一个长的DNA-RNA杂交体，并从模板中取代未转录的DNA，产生一个特征性的分裂末端结构。DNA-RNA杂交体也在RNA聚合酶II从缺乏3'寡聚体（dC）尾的双链体DNA模板转录期间形成，其主要发生在单链断裂或末端。因此，小牛胸腺RNA聚合酶II在体外进行的转录延伸反应在几个方面不同于必须在体内发生的反应。
Addition of 10 to 100 oligodeoxycytidylate residues to the 3'-OH termini of T7 bacteriophage DNA produces a highly efficient template for transcription in vitro with purified calf thymus RNA polymerase II. Transcription initiates rapidly and selectively at the oligo (dC) ends of such a template and essentially all of the active RNA polymerase II molecules are then committed to a long period of RNA chain elongation. This allows the direct study of the RNA chain elongation and termination reactions and also permits determination of the concentration of active RNA polymerase II that is present. From 15 to 25% of the RNA polymerase molecules in our current preparations are active in these reactions. RNA chain elongation by calf thymus RNA polymerase II is relatively slow (7 nucleotides/s) even at saturating substrate concentrations. The in vitro elongation process appears to be discontinuous, with elongating polymerase molecules pausing for significant periods at certain sequences along the DNA. There is a low, but measurable frequency of RNA chain termination at some sites; however, the majority of elongating transcripts can grow to large sizes (over 6000 nucleotides). Surprisingly, over 60% of the active calf thymus RNA polymerase II molecules form a long DNA-RNA hybrid during in vitro transcription and displace the nontranscribed DNA from the template to produce a characteristic split end structure. DNA-RNA hybrids are also formed during transcription by RNA polymerase II from duplex DNA templates lacking 3' oligo(dC) tails, which takes place predominantly at single strand breaks or ends. Thus the transcriptional elongation reaction carried out by calf thymus RNA polymerase II in vitro differs in several respects from that which must take place in vivo.