Dynamic and structural characterisation of multiple steps during complex formation between E. coli RNA polymerase and the tetR promoter from pSC101.

Abstract

Kinetic, functional and structural studies of the recognition of the tetR promoter from pSC101 by E. coli RNA polymerase allowed the characterization of several steps in the specific complex formation and transcription initiation process. First, enzyme and DNA enter in a short life-time complex. An isomerization will convert this unstable complex into a closed stable one where RNA polymerase is tightly attached without establishing stable chemical contacts with the bases. In the next step, stable close contacts appear between both macromolecules involving mainly the downstream part of the promoter. A further isomerization will lead to an open complex where DNA is locally melted and the system is able to initiate transcription. This latter process is accompanied by changes in the upstream part of the promoter. Finally, in vitro transcription assays showed that the position of the major transcription start sites depends on temperature. From the reported results, it appears that the recognition event is a sequential process where different structural elements of the promoter, that can be located apart in the sequence, are involved in a concerted manner in each stage.