9bvt

From Proteopedia

RNA Pol II - High Mn(+2) concentration

Structural highlights

9bvt is a 10 chain structure with sequence from Saccharomyces cerevisiae. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 3.4Å
Ligands:	, ,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

RPB1_YEAST DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. Largest and catalytic component of RNA polymerase II which synthesizes mRNA precursors and many functional non-coding RNAs. Forms the polymerase active center together with the second largest subunit. Pol II is the central component of the basal RNA polymerase II transcription machinery. During a transcription cycle, Pol II, general transcription factors and the Mediator complex assemble as the preinitiation complex (PIC) at the promoter. 11-15 base pairs of DNA surrounding the transcription start site are melted and the single stranded DNA template strand of the promoter is positioned deeply within the central active site cleft of Pol II to form the open complex. After synthesis of about 30 bases of RNA, Pol II releases its contacts with the core promoter and the rest of the transcription machinery (promoter clearance) and enters the stage of transcription elongation in which it moves on the template as the transcript elongates. Pol II appears to oscillate between inactive and active conformations at each step of nucleotide addition. Elongation is influenced by the phosphorylation status of the C-terminal domain (CTD) of Pol II largest subunit (RPB1), which serves as a platform for assembly of factors that regulate transcription initiation, elongation, termination and mRNA processing. Pol II is composed of mobile elements that move relative to each other. The core element with the central large cleft comprises RPB3, RBP10, RPB11, RPB12 and regions of RPB1 and RPB2 forming the active center. The clamp element (portions of RPB1, RPB2 and RPB3) is connected to the core through a set of flexible switches and moves to open and close the cleft. A bridging helix emanates from RPB1 and crosses the cleft near the catalytic site and is thought to promote translocation of Pol II by acting as a ratchet that moves the RNA-DNA hybrid through the active site by switching from straight to bent conformations at each step of nucleotide addition. In elongating Pol II, the lid loop (RPB1) appears to act as a wedge to drive apart the DNA and RNA strands at the upstream end of the transcription bubble and guide the RNA strand toward the RNA exit groove located near the base of the largely unstructured CTD domain of RPB1. The rudder loop (RPB1) interacts with single stranded DNA after separation from the RNA strand, likely preventing reassociation with the exiting RNA. The cleft is surrounded by jaws: an upper jaw formed by portions of RBP1, RPB2 and RPB9, and a lower jaw, formed by RPB5 and portions of RBP1. The jaws are thought to grab the incoming DNA template, mainly by RPB5 direct contacts to DNA.

Publication Abstract from PubMed

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(2+), and, more importantly, a putative third (site C) Mg(2+) 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(2+) 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.

Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free-electron laser.,Lin G, Barnes CO, Weiss S, Dutagaci B, Qiu C, Feig M, Song J, Lyubimov A, Cohen AE, Kaplan CD, Calero G Proc Natl Acad Sci U S A. 2024 Sep 3;121(36):e2318527121. doi: , 10.1073/pnas.2318527121. Epub 2024 Aug 27. PMID:39190355^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Lin G, Barnes CO, Weiss S, Dutagaci B, Qiu C, Feig M, Song J, Lyubimov A, Cohen AE, Kaplan CD, Calero G. Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free-electron laser. Proc Natl Acad Sci U S A. 2024 Sep 3;121(36):e2318527121. PMID:39190355 doi:10.1073/pnas.2318527121