Intrinsic cleavage of RNA polymerase II adopts a nucleobase-independent mechanism assisted by transcript phosphate
AuthorsTse, Carmen Ka Man
Sheong, Fu Kit
Chow, Hoi Yee
Cheung, Peter Pak-Hang
KAUST DepartmentComputational Bioscience Research Center (CBRC)
Computer Science Program
Computer, Electrical and Mathematical Sciences and Engineering (CEMSE) Division
Structural and Functional Bioinformatics Group
KAUST Grant NumberOSR-2016-CRG5-3007
Online Publication Date2019-02-11
Print Publication Date2019-03
Permanent link to this recordhttp://hdl.handle.net/10754/631390
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AbstractRNA polymerase II (Pol II) utilizes the same active site for polymerization and intrinsic cleavage. Pol II proofreads the nascent transcript via its intrinsic nuclease activity to maintain high transcriptional fidelity critical for cell growth and viability. The detailed catalytic mechanism of intrinsic cleavage remains unknown. Here, we combined ab initio quantum mechanics/molecular mechanics studies and biochemical cleavage assays to show that Pol II utilizes downstream phosphate oxygen to activate the attacking nucleophile in hydrolysis, while the newly formed 3′-end is protonated through active-site water without a defined general acid. Experimentally, alteration of downstream phosphate oxygen either by 2′-5′ sugar linkage or stereo-specific thio-substitution of phosphate oxygen drastically reduced cleavage rate. We showed by N7-modification that guanine nucleobase is not directly involved as an acid–base catalyst. Our proposed mechanism provides important insights into the intrinsic transcriptional cleavage reaction, an essential step in transcriptional fidelity control.
CitationTse CKM, Xu J, Xu L, Sheong FK, Wang S, et al. (2019) Intrinsic cleavage of RNA polymerase II adopts a nucleobase-independent mechanism assisted by transcript phosphate. Nature Catalysis. Available: http://dx.doi.org/10.1038/s41929-019-0227-5.
SponsorsWe thank Z. Lin for helpful discussions. This work was supported by the Hong Kong Research Grant Council (grant nos. HKUST C6009-15G and AoE/P-705/16 to X.H. and X.L.; 16302214 and T31-605/18-W to X.H.), the King Abdullah University of Science and Technology Office of Sponsored Research (OSR) (OSR-2016-CRG5-3007 to X.H. and X.G.), the Shenzhen Science and Technology Innovation Committee (JCYJ20170413173837121 to X.H.), the Innovation and Technology Commission (ITC-CNERC14SC01 to X.H.), and the National Institutes of Health (grant no. R35-GM127040 to Y.Z.; grant no. GM102362 to D.W.). X.H. is the Padma Harilela Associate Professor of Science. This research made use of the computing resources of the Supercomputing Laboratory at King Abdullah University of Science and Technology.