8U9R

STRUCTURAL BASIS OF TRANSCRIPTION: RNA POLYMERASE II SUBSTRATE BINDING AND METAL COORDINATION USING A FREE-ELECTRON LASER

Summary for 8U9R

• Entry DOI: 10.2210/pdb8u9r/pdb
• Descriptor: DNA-directed RNA polymerase II subunit RPB1, DNA-directed RNA polymerases II subunit RPABC5, DNA-directed RNA polymerase II subunit RPB11, ... (19 entities in total)
• Functional Keywords: transcription, multiprotein complex, free-electron laser, molecular machine
• Biological source: Saccharomyces cerevisiae (baker's yeast); More
• Total number of polymer chains: 14
• Total formula weight: 522810.44

Authors

Arjunan, P.,Calero, G.,Kaplan, C.D. (deposition date: 2023-09-20, release date: 2024-09-18, Last modification date: 2024-11-13)

Primary citation

Lin, G.,Barnes, C.O.,Weiss, S.,Dutagaci, B.,Qiu, C.,Feig, M.,Song, J.,Lyubimov, A.,Cohen, A.E.,Kaplan, C.D.,Calero, G.
Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free-electron laser.
Proc.Natl.Acad.Sci.USA, 121:e2318527121-e2318527121, 2024

PubMed Abstract: Catalysis and translocation of multisubunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near-atomic resolution and precise arrangement of key active site components have been elusive. Here, we present the free-electron laser (FEL) structures of a matched ATP-bound Pol II and the hyperactive Rpb1 T834P bridge helix (BH) mutant at the highest resolution to date. The radiation-damage-free FEL structures reveal the full active site interaction network, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg, and, more importantly, a putative third (site C) Mg analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structures indicate that the third Mg is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/BH interactions induce conformational changes that could allow translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the T834P mutant reveal rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

PubMed: 39190355
DOI: 10.1073/pnas.2318527121

Experimental method

X-RAY DIFFRACTION (3.34 Å)