气候变化领域集成服务门户(CCPortal): Obligate movements of an active site-linked surface domain control RNA polymerase elongation and pausing via a Phe pocket anchor

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DOI	10.1073/pnas.2101805118
	Obligate movements of an active site-linked surface domain control RNA polymerase elongation and pausing via a Phe pocket anchor
	Bao Y.; Landick R.
发表日期	2021
ISSN	0027-8424
卷号	118 期号:36
英文摘要	The catalytic trigger loop (TL) in RNA polymerase (RNAP) alternates between unstructured and helical hairpin conformations to admit and then contact the NTP substrate during transcription. In many bacterial lineages, the TL is interrupted by insertions of two to five surface-exposed, sandwich-barrel hybrid motifs (SBHMs) of poorly understood function. The 188-amino acid, two-SBHM insertion in Escherichia coli RNAP, called SI3, occupies different locations in elongating, NTP-bound, and paused transcription complexes, but its dynamics during active transcription and pausing are undefined. Here, we report the design, optimization, and use of a Cys-triplet reporter to measure the positional bias of SI3 in different transcription complexes and to determine the effect of restricting SI3 movement on nucleotide addition and pausing. We describe the use of H2O2 as a superior oxidant for RNAP disulfide reporters. NTP binding biases SI3 toward the closed conformation, whereas transcriptional pausing biases SI3 toward a swiveled position that inhibits TL folding. We find that SI3 must change location in every round of nucleotide addition and that restricting its movements inhibits both transcript elongation and pausing. These dynamics are modulated by a crucial Phe pocket formed by the junction of the two SBHM domains. This SI3 Phe pocket captures a Phe residue in the RNAP jaw when the TL unfolds, explaining the similar phenotypes of alterations in the jaw and SI3. Our findings establish that SI3 functions by modulating TL folding to aid transcriptional regulation and to reset secondary channel trafficking in every round of nucleotide addition. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Catalysis; Pausing; Regulation; RNA polymerase; Transcription
语种	英语
scopus关键词	phenylalanine; RNA polymerase; Article; enzyme active site; enzyme regulation; Escherichia coli; genetic transcription; nonhuman; protein folding; protein unfolding; RNA translation; trigger loop
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/238810
作者单位	Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, United States; Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, United States
推荐引用方式 GB/T 7714	Bao Y.,Landick R.. Obligate movements of an active site-linked surface domain control RNA polymerase elongation and pausing via a Phe pocket anchor[J],2021,118(36).
APA	Bao Y.,&Landick R..(2021).Obligate movements of an active site-linked surface domain control RNA polymerase elongation and pausing via a Phe pocket anchor.Proceedings of the National Academy of Sciences of the United States of America,118(36).
MLA	Bao Y.,et al."Obligate movements of an active site-linked surface domain control RNA polymerase elongation and pausing via a Phe pocket anchor".Proceedings of the National Academy of Sciences of the United States of America 118.36(2021).