6uq3

From Proteopedia

RNA polymerase II elongation complex with 5-guanidinohydantoin lesion in state 5

PDB ID 6uq3

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Structural highlights

6uq3 is a 10 chain structure with sequence from Saccharomyces cerevisiae S288C and Synthetic construct. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 3.47Å
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

Oxidation of guanine generates several types of DNA lesions, such as 8-oxoguanine (8OG), 5-guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp). These guanine-derived oxidative DNA lesions interfere with both replication and transcription. However, the molecular mechanism of transcription processing of Gh and Sp remains unknown. In this study, by combining biochemical and structural analysis, we revealed distinct transcriptional processing of these chemically related oxidized lesions: 8OG allows both error-free and error-prone bypass, whereas Gh or Sp causes strong stalling and only allows slow error-prone incorporation of purines. Our structural studies provide snapshots of how polymerase II (Pol II) is stalled by a nonbulky Gh lesion in a stepwise manner, including the initial lesion encounter, ATP binding, ATP incorporation, jammed translocation, and arrested states. We show that while Gh can form hydrogen bonds with adenosine monophosphate (AMP) during incorporation, this base pair hydrogen bonding is not sufficient to hold an ATP substrate in the addition site and is not stable during Pol II translocation after the chemistry step. Intriguingly, we reveal a unique structural reconfiguration of the Gh lesion in which the hydantoin ring rotates approximately 90 degrees and is perpendicular to the upstream base pair planes. The perpendicular hydantoin ring of Gh is stabilized by noncanonical lone pair-pi and CH-pi interactions, as well as hydrogen bonds. As a result, the Gh lesion, as a functional mimic of a 1,2-intrastrand crosslink, occupies canonical -1 and +1 template positions and compromises the loading of the downstream template base. Furthermore, we suggest Gh and Sp lesions are potential targets of transcription-coupled repair.

RNA polymerase II stalls on oxidative DNA damage via a torsion-latch mechanism involving lone pair-pi and CH-pi interactions.,Oh J, Fleming AM, Xu J, Chong J, Burrows CJ, Wang D Proc Natl Acad Sci U S A. 2020 Apr 28;117(17):9338-9348. doi:, 10.1073/pnas.1919904117. Epub 2020 Apr 13. PMID:32284409^[1]

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

References

↑ Oh J, Fleming AM, Xu J, Chong J, Burrows CJ, Wang D. RNA polymerase II stalls on oxidative DNA damage via a torsion-latch mechanism involving lone pair-π and CH-π interactions. Proc Natl Acad Sci U S A. 2020 Apr 28;117(17):9338-9348. PMID:32284409 doi:10.1073/pnas.1919904117