Promoter escape by RNA polymerase, the transition between the initiation and elongation, is a critical step that defines transcription output at many promoters. In the present study we used a real-time fluorescence assay for promoter melting and escape to study the determinants of the escape. Perturbation of core promoter–polymerase contacts had opposing effects on the rates of melting and escape, demonstrating a direct role of core promoter elements sequence in setting not only the kinetics of promoter melting, but also the kinetics of promoter escape. The start of RNA synthesis is accompanied by an enlargement of the transcription bubble and pulling in of the downstream DNA into the enzyme, resulting in DNA scrunching. Promoter escape results in collapse of the enlarged bubble. To test whether the energy that could be potentially released by the collapse of the bubble plays a role in determining escape kinetics, we measured the rates of promoter escape in promoter constructs, in which the amount of this energy was perturbed by introducing sequence mismatches. We found no significant changes in the rate of promoter escape with these promoter constructs suggesting that the energy released upon bubble collapse does not play a critical role in determining the kinetics of promoter escape.

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