Structural origins of Escherichia coli RNA polymerase open promoter complex stability

被引:21
作者
Saecker, Ruth M. [1 ]
Chen, James [1 ,4 ]
Chiu, Courtney E. [1 ]
Malone, Brandon [1 ]
Sotiris, Johanna [2 ]
Ebrahim, Mark [2 ]
Yen, Laura Y. [3 ,5 ]
Eng, Edward T. [3 ]
Darst, Seth A. [1 ]
机构
[1] Rockefeller Univ, Lab Mol Biophys, New York, NY 10065 USA
[2] Rockefeller Univ, Evelyn Gruss Lipper Cryo Electron Microscopy Reso, New York, NY 10065 USA
[3] New York Struct Biol Ctr, Simons Electron Microscopy Ctr, Natl Resource Automated Mol Microscopy, New York, NY 10027 USA
[4] New York Univ, Grossman Sch Med, Dept Cell Biol, New York, NY 10016 USA
[5] Columbia Univ, Vagelos Coll Phys & Surg, Dept Physiol & Cellular Biophys, New York, NY 10032 USA
关键词
transcription; RNA polymerase; cryo-EM; open complex; promoter DNA; P-R PROMOTER; TRANSCRIPTION INITIATION; CONFORMATIONAL-CHANGES; ABORTIVE INITIATION; BRANCHED PATHWAY; FINE-STRUCTURE; IN-VITRO; DNA; RECOGNITION; ESCAPE;
D O I
10.1073/pnas.2112877118
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms "open" complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 degrees C: lambda P-R (t(1/2) similar to 10 h), T7A1 (t(1/2) similar to 4 min), and a point mutant in lambda P-R (lambda PR-5C) (t(1/2) similar to 2 h). Two distinct RPo conformers are populated at lambda P-R, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand "scrunches" inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.
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页数:9
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