Major Conformational Changes During T7RNAP Transcription Initiation Coincide with, and are Required for, Promoter Release

During transcription initiation conformational changes in the transcriptional machinery are required to accommodate the growing RNA, to allow the polymerase to release the promoter, and to endow the elongation complex with high processivity. In T7 RNA polymerase these changes involve refolding and reorientation of elements of the N-terminal domain, as well as changes in how the DNA is bound within the complex. However, when and where these conformational changes occur is unknown, and the role of these changes in allowing the polymerase to disengage the promoter is poorly understood. To address this we have used chemical nucleases tethered to the polymerase to monitor conformational changes, and engineered disulfide bonds to block conformational changes at defined steps in transcription. We find that many of the major structural transitions occur cooperatively, at a point coincident with promoter release. Moreover, promoter release requires that two elements of the polymerase which form a continuous promoter recognition surface in the initial transcription complex move apart: if this movement is blocked the polymerase cannot disengage the promoter.