Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy
During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAP), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, time-resolved cryo-elec...
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Published in | bioRxiv |
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Main Authors | , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
14.03.2024
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Online Access | Get full text |
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Summary: | During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAP), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, time-resolved cryo-electron microscopy (cryo-EM) was used to capture four intermediates populated 120 or 500 milliseconds (ms) after mixing
σ
-RNAP and the λP
promoter. Cryo-EM snapshots revealed the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As nt-strand "read-out" extends, the RNAP clamp closes, expelling an inhibitory σ
domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating yet unknown conformational changes load it in subsequent steps. Because these events likely describe DNA opening at many bacterial promoters, this study provides needed insights into how DNA sequence regulates steps of RPo formation. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Working Paper/Pre-Print-1 content type line 23 |
ISSN: | 2692-8205 2692-8205 |
DOI: | 10.1101/2024.03.13.584744 |