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Structure paper

TitleEarly intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.
Journal, issue, pagesNat Struct Mol Biol, Vol. 31, Issue 11, Page 1778-1788, Year 2024
Publish dateJul 1, 2024
AuthorsRuth M Saecker / Andreas U Mueller / Brandon Malone / James Chen / William C Budell / Venkata P Dandey / Kashyap Maruthi / Joshua H Mendez / Nina Molina / Edward T Eng / Laura Y Yen / Clinton S Potter / Bridget Carragher / Seth A Darst /
PubMed AbstractDuring formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural ...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-RNAP and the λP 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 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.
External linksNat Struct Mol Biol / PubMed:38951624
MethodsEM (single particle)
Resolution2.8 - 3.1 Å
Structure data

41433

8to1

EMDB-41433, PDB-8to1:
Escherichia coli RNA polymerase unwinding intermediate (I1a) at the lambda PR promoter
Method: EM (single particle) / Resolution: 2.8 Å

41437

8to6

EMDB-41437, PDB-8to6:
Escherichia coli RNA polymerase unwinding intermediate (I1d) at the lambda PR promoter
Method: EM (single particle) / Resolution: 2.9 Å

41439

8to8

EMDB-41439, PDB-8to8:
Escherichia coli RNA polymerase unwinding intermediate (I1b) at the lambda PR promoter
Method: EM (single particle) / Resolution: 2.9 Å

41448

8toe

EMDB-41448, PDB-8toe:
Escherichia coli RNA polymerase unwinding intermediate (I1c) at the lambda PR promoter
Method: EM (single particle) / Resolution: 2.9 Å

41456

8tom

EMDB-41456, PDB-8tom:
Escherichia coli RNA polymerase closed complex intermediate at the lambda PR promoter
Method: EM (single particle) / Resolution: 3.1 Å

Chemicals

ChemComp-4QM:
(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-10,13-dimethyl-17-[(2R)-pentan-2-yl]-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-triol

ChemComp-MG:
Unknown entry

ChemComp-ZN:
Unknown entry

ChemComp-1N7:
CHAPSO / detergent*YM

Source
  • Escherichia coli (E. coli)
  • escherichia coli (strain k12) (bacteria)
  • escherichia phage lambda (virus)
  • escherichia coli k-12 (bacteria)
Keywordstranscription/DNA / DNA-dependent RNA polymerase / transcription / intermediate / DNA promoter / DNA unwinding / transcription-DNA complex

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