Bateman E, Paule M R
Department of Biochemistry, Colorado State University, Fort Collins 80523.
Mol Cell Biol. 1988 May;8(5):1940-6. doi: 10.1128/mcb.8.5.1940-1946.1988.
Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.
在棘阿米巴原虫rRNA转录起始因子（TIF）和RNA聚合酶I与DNA形成的复合物进行转录起始和延伸过程中，开展了化学足迹分析和拓扑分析。结果表明，TIF和聚合酶与启动子的结合不会改变DNA模板的超螺旋结构，并且模板对焦碳酸二乙酯修饰不敏感，焦碳酸二乙酯可识别解链的DNA区域。因此，与细菌RNA聚合酶不同，真核生物RNA聚合酶I-启动子复合物在体外添加核苷酸之前处于封闭构象。RNA聚合酶I引发并进行3'-O-甲基CTP限制的转位会导致聚合酶-TIF足迹分离，而TIF足迹保持不变。相比之下，引发和转位会导致聚合酶-DNA复合物的构象发生显著变化，最终形成至少10个碱基对的解链DNA区域。
