Brodolin K, Buckle M
Laboratory of Molecular Genetics of Microorganisms, Institute of Molecular Genetics, Kurchatov Sq. 2, Moscow 123182, Russia.
J Mol Biol. 2001 Mar 16;307(1):25-30. doi: 10.1006/jmbi.2000.4483.
Formaldehyde cross-linking was used in a kinetic analysis of RNA polymerase-lacUV5 promoter interactions in open complexes (RP(o)). RP(o) quenched from 37 degrees C to 14 degrees C isomerised to a closed, competitor resistant, complex (RP(LT)). We observed that contacts of the beta' and sigma subunits with the positions -3, -5 of the non-template DNA strand disappeared very quickly during the first 30 seconds after the temperature downshift. The re-annealing of the DNA downstream of the transcription start site takes place in the same time scale. However re-annealing of the upstream part of the transcription bubble was slower and completed within five minutes. The results support a two-step model of promoter melting and suggest that conformational changes in the RNA polymerase occur concurrently with the melting around the transcription start site.
甲醛交联被用于对开放复合物（RP(o)）中RNA聚合酶与lacUV5启动子相互作用的动力学分析。从37℃淬灭至14℃的RP(o)异构化为一种封闭的、抗竞争剂的复合物（RP(LT)）。我们观察到，在温度下降后的最初30秒内，β'亚基和σ亚基与非模板DNA链-3、-5位置的接触迅速消失。转录起始位点下游DNA的重新退火在相同的时间尺度内发生。然而，转录泡上游部分的重新退火较慢，在五分钟内完成。这些结果支持启动子解链的两步模型，并表明RNA聚合酶的构象变化与转录起始位点周围的解链同时发生。
