Mishoe H, Brady J N, Lancz G, Salzman N P
J Biol Chem. 1984 Feb 25;259(4):2236-42.
We have mapped a major initiation site of purified calf thymus RNA polymerase II in the cloned adenovirus 2 major late promoter. The specificity of this initiation site has been determined by "run-off" transcriptional analysis and by RNase T1 analysis which employs single-stranded M13 phage DNA containing the Adenovirus 2 major late promoter as probe. The TATAAA region which is used as the start site by the purified RNA polymerase II for in vitro transcription is 30 base pairs upstream from the adenovirus major late in vivo start site. The exact sequence also exists at two sites within the pBR322 plasmid but initiation does not occur at either of these sites. This indicates that the purified enzyme is not just recognizing AT-rich regions but that it is recognizing both the TATA box and its surrounding sequences. The purified RNA polymerase II transcriptional initiation site is used when transcription was carried out on either a superhelical (FI) or linear (FIII) DNA template. Selective initiation of transcription on FI DNA required (NH4)2SO4 concentrations which ranged from 90 to 150 mM. In contrast, selective initiation of transcription on FIII DNA was observed at (NH4)2SO4 concentrations that ranged from 30 to 120 mM.
我们已在克隆的腺病毒2型主要晚期启动子中定位了纯化的小牛胸腺RNA聚合酶II的一个主要起始位点。该起始位点的特异性已通过“连续”转录分析以及核糖核酸酶T1分析得以确定，核糖核酸酶T1分析采用含有腺病毒2型主要晚期启动子的单链M13噬菌体DNA作为探针。纯化的RNA聚合酶II用于体外转录的起始位点TATAAA区域，位于腺病毒主要晚期体内起始位点上游30个碱基对处。pBR322质粒内的两个位点也存在完全相同的序列，但在这两个位点均未发生起始。这表明纯化的酶并非仅仅识别富含AT的区域，而是识别TATA框及其周围序列。当在超螺旋（FI）或线性（FIII）DNA模板上进行转录时，会使用纯化的RNA聚合酶II转录起始位点。在FI DNA上进行选择性转录起始所需的硫酸铵浓度范围为90至150 mM。相比之下，在硫酸铵浓度范围为30至120 mM时，可观察到在FIII DNA上的选择性转录起始。
