Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 102, Issue 17, Pages 5958-5963Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0407141102
Keywords
abortive transcription; in vitro RNA synthesis
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Like multisubunit RNA polymerases (RNAPs), T7 RNAP frequently releases its transcript over the initial 8-12 transcribed nucleotides, when it still contacts the promoter. This abortive cycling, which is most prominent with initial sequences that deviate from those of T7 late genes, eventually compromises productive transcription. Starting from an in vivo situation where transcription of a target gene by T7 RNAP is virtually abolished because of extensive abortive cycling, we have selected a mutation in RNAP that restores target gene expression. In vitro, this mutation (P266L) weakens promoter binding but markedly reduces abortive cycling over a variety of initial sequences by stabilizing the transcription complex at nucleotides 5-8. Other substitutions of P266 have similar effects. X-ray data show that during the transition from initial to elongation complex, the N-terminal region undergoes a major structural switch of which P266 constitutes one of the hinges. How the mutation might facilitate this switch is tentatively discussed. On the practical side, the mutation can significantly improve in vitro transcription, particularly from templates carrying unfavorable initial sequences.
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