Kulbachinskiy A V, Nikiforov V G, Brodolin K L
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182, Russia.
Biochemistry (Mosc). 2005 Nov;70(11):1227-30. doi: 10.1007/s10541-005-0251-8.
The interaction of RNA polymerases from Escherichia coli and Thermus aquaticus with lacUV5 promoter was studied at various temperatures. Using DNA-protein cross-linking induced by formaldehyde, it was demonstrated that each RNA polymerase formed a unique pattern of contacts with DNA in the open promoter complex. In the case of E. coli RNA polymerase, beta and sigma subunits were involved into formation of cross-links with the promoter, whereas in the case of T. aquaticus RNA polymerase its beta subunit formed the cross-links with the promoter. A cross-linking pattern in promoter complexes of a hybrid holoenzyme comprised of the core-enzyme of E. coli and sigma subunit of T. aquaticus was similar to that of the E. coli holoenzyme. This suggests that DNA-protein contacts in the promoter complex are primarily determined by the core-enzyme of RNA polymerase. However, temperature-dependent behavior of contact formation is determined by the sigma subunit. Results of the present study indicate that the method of formaldehyde cross-linking can be employed for elucidation of differences in the structure of promoter complexes of RNA polymerases from various bacteria.
在不同温度下研究了来自大肠杆菌和嗜热水生栖热菌的RNA聚合酶与lacUV5启动子的相互作用。利用甲醛诱导的DNA-蛋白质交联，证明了每种RNA聚合酶在开放启动子复合物中与DNA形成了独特的接触模式。对于大肠杆菌RNA聚合酶，β亚基和σ亚基参与与启动子形成交联，而对于嗜热水生栖热菌RNA聚合酶，其β亚基与启动子形成交联。由大肠杆菌核心酶和嗜热水生栖热菌σ亚基组成的杂交全酶在启动子复合物中的交联模式与大肠杆菌全酶相似。这表明启动子复合物中的DNA-蛋白质接触主要由RNA聚合酶的核心酶决定。然而，接触形成的温度依赖性行为由σ亚基决定。本研究结果表明，甲醛交联方法可用于阐明来自不同细菌的RNA聚合酶启动子复合物结构的差异。
