Evidence for DNA bending at the T7 RNA polymerase promoter

Abstract

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.