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Molecular and Cellular Biology, February 2002, p. 762-773, Vol. 22, No. 3
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.3.762-773.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Translocation after Synthesis of a Four-Nucleotide RNA Commits RNA Polymerase II to Promoter Escape

Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215

Received 7 June 2001/ Returned for modification 10 July 2001/ Accepted 29 October 2001

Transcription is a complex process, the regulation of which is crucial for cellular and organismic growth and development. Deciphering the molecular mechanisms that define transcription is essential to understanding the regulation of RNA synthesis. Here we describe the molecular mechanism of escape commitment, a critical step in early RNA polymerase II transcription. During escape commitment ternary transcribing complexes become stable and committed to proceeding forward through promoter escape and the remainder of the transcription reaction. We found that the point in the transcription reaction at which escape commitment occurs depends on the length of the transcript RNA (4 nucleotides [nt]) as opposed to the position of the active site of the polymerase with respect to promoter DNA elements. We found that single-stranded nucleic acids can inhibit escape commitment, and we identified oligonucleotides that are potent inhibitors of this specific step. These inhibitors bind RNA polymerase II with low nanomolar affinity and sequence specificity, and they block both promoter-dependent and promoter-independent transcription, the latter occurring in the absence of general transcription factors. We demonstrate that escape commitment involves translocation of the RNA polymerase II active site between synthesis of the third and fourth phosphodiester bonds. We propose that a conformational change in ternary transcription complexes occurs during translocation after synthesis of a 4-nt RNA to render complexes escape committed.

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