Brodolin K, Mustaev A, Severinov K, Nikiforov V
Institute of Molecular Genetics Russian Academy of Sciences, Kurchatov Sq. 46, Moscow 123182, Russia.
J Biol Chem. 2000 Feb 4;275(5):3661-6. doi: 10.1074/jbc.275.5.3661.
Identification of the RNA polymerase functional regions involved in interactions with promoter is a basis for understanding the mechanism of transcription initiation. We have used formaldehyde cross-linking to identify a region of Escherichia coli RNA polymerase beta' subunit contacting lacUV5 promoter in open complex. Treatment of open complex with formaldehyde results in cross-linking of beta' and sigma(70) subunits at positions -5 and -3 on the nontemplate strand of the promoter DNA. These cross-links reflect specific interactions between RNA polymerase and promoter established in open complex. The positions of formaldehyde cross-links in the beta' subunit were mapped to the N-terminal segment (Cys(198)-Met(237)), which is contiguous to the evolutionary conserved region B. The proximity of the beta' and sigma cross-links suggest that the N-terminal region of the beta' subunit, interacting with single-stranded promoter DNA, can cooperate with the sigma subunit in the process of open complex formation.
鉴定参与与启动子相互作用的RNA聚合酶功能区域是理解转录起始机制的基础。我们利用甲醛交联来鉴定大肠杆菌RNA聚合酶β'亚基在开放复合物中与lacUV5启动子接触的区域。用甲醛处理开放复合物会导致β'亚基和σ（70）亚基在启动子DNA非模板链上的-5和-3位置发生交联。这些交联反映了在开放复合物中RNA聚合酶与启动子之间建立的特异性相互作用。β'亚基中甲醛交联的位置被定位到N端片段（Cys（198）-Met（237）），该片段与进化保守区域B相邻。β'亚基和σ亚基交联的接近性表明，与单链启动子DNA相互作用的β'亚基N端区域在开放复合物形成过程中可与σ亚基协同作用。
