Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.

Autor: Saecker RM; Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY, USA., Mueller AU; Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY, USA., Malone B; Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY, USA.; Memorial Sloan Kettering Cancer Center, Sloan Kettering Institute, New York, NY, USA., Chen J; Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY, USA.; Laboratory of Host-Pathogen Biology, The Rockefeller University, New York, NY, USA., Budell WC; The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA., Dandey VP; The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA.; National Institute of Environmental Health Sciences, Durham, NC, USA., Maruthi K; The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA., Mendez JH; The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA., Molina N; Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY, USA., Eng ET; The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA., Yen LY; The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA., Potter CS; The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA.; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.; Chan Zuckerberg Imaging Institute, San Francisco, CA, USA., Carragher B; The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA.; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.; Chan Zuckerberg Imaging Institute, San Francisco, CA, USA., Darst SA; Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY, USA. darst@rockefeller.edu.
Jazyk: angličtina
Zdroj: Nature structural & molecular biology [Nat Struct Mol Biol] 2024 Nov; Vol. 31 (11), pp. 1778-1788. Date of Electronic Publication: 2024 Jul 01.
DOI: 10.1038/s41594-024-01349-9
Abstrakt: During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ 70 -RNAP and the λP R promoter. Cryo-EM snapshots revealed that 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 the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory σ 70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.
Competing Interests: Competing interests The authors declare there are no competing interests.
(© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)
Databáze: MEDLINE
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