期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：3
页码：743-748
DOI：10.1073/pnas.1421067112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceRNA polymerase is a vital enzyme responsible for the first step in gene expression. Despite extensive studies, fundamental questions about its kinetic and mechanistic properties still remain unanswered. The trigger loop is a conserved domain within RNA polymerase that has been linked to the enzyme's average elongation velocity and pausing behavior. In this study, we use optical tweezers, a single molecule technique, to analyze the behavior of two mutant polymerases with a single point mutation in their trigger loop domain and compare it to the WT. By looking at individual enzymes we are able to separate continuous elongation from pausing and create a kinetic and mechanistic model in which trigger loop folding-unfolding dynamics controls transcription elongation. Two components of the RNA polymerase (RNAP) catalytic center, the bridge helix and the trigger loop (TL), have been linked with changes in elongation rate and pausing. Here, single molecule experiments with the WT and two TL-tip mutants of the Escherichia coli enzyme reveal that tip mutations modulate RNAP's pause-free velocity, identifying TL conformational changes as one of two rate-determining steps in elongation. Consistent with this observation, we find a direct correlation between helix propensity of the modified amino acid and pause-free velocity. Moreover, nucleotide analogs affect transcription rate, suggesting that their binding energy also influences TL folding. A kinetic model in which elongation occurs in two steps, TL folding on nucleoside triphosphate (NTP) binding followed by NTP incorporation/pyrophosphate release, quantitatively accounts for these results. The TL plays no role in pause recovery remaining unfolded during a pause. This model suggests a finely tuned mechanism that balances transcription speed and fidelity.
