Ujvári A, Martin C T
Department of Chemistry, University of Massachusetts, Amherst, MA, 01003-4510, USA.
J Mol Biol. 2000 Feb 4;295(5):1173-84. doi: 10.1006/jmbi.1999.3418.
Phage T7 RNA polymerase is the only DNA-dependent RNA polymerase for which we have a high-resolution structure of the promoter-bound complex. Recent studies with the more complex RNA polymerases have suggested a role for DNA wrapping in the initiation of transcription. Here, circular permutation gel retardation assays provide evidence that the polymerase does indeed bend its promoter DNA. A complementary set of experiments employing differential phasing from an array of phased A-tracts provides further evidence for both intrinsic and polymerase-induced bends in the T7 RNA polymerase promoter DNA. The bend in the complex is predicted to be about 40-60 degrees and to be centered around positions -2 to +1, at the start site for transcription, while the intrinsic bend is much smaller (about 10 degrees ). These results, viewed in the light of a recent crystal structure for the complex, suggest a mechanism by which binding leads directly to bending. Bending at the start site would then facilitate the melting necessary to initiate transcription.
T7噬菌体RNA聚合酶是我们拥有其启动子结合复合物高分辨率结构的唯一一种依赖DNA的RNA聚合酶。最近对更复杂的RNA聚合酶的研究表明，DNA缠绕在转录起始过程中发挥作用。在此，环形置换凝胶阻滞试验提供了证据，证明该聚合酶确实会使其启动子DNA弯曲。一组互补的实验采用来自一系列相位A序列的差异相位，为T7 RNA聚合酶启动子DNA中的固有弯曲和聚合酶诱导的弯曲提供了进一步证据。复合物中的弯曲预计约为40 - 60度，以转录起始位点的-2至+1位置为中心，而固有弯曲要小得多（约10度）。根据该复合物最近的晶体结构来看，这些结果提示了一种结合直接导致弯曲的机制。起始位点处的弯曲随后将促进转录起始所需的解链。
