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      Intrinsic Cleavage of RNA Polymerase II Adopts a Nucleobase-independent Mechanism Assisted by Transcript Phosphate

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          Abstract

          RNA polymerase II (Pol II) utilises the same active site for polymerization and intrinsic cleavage. Pol II proofreads the nascent transcript by 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 utilises 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 does not directly involve as acid-base catalyst. Our proposed mechanism provides important insights into the understanding of intrinsic transcriptional cleavage reaction, an essential step of transcriptional fidelity control.

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          Most cited references 50

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                Author and article information

                Journal
                101734042
                48010
                Nat Energy
                Nat Energy
                Nature energy
                2058-7546
                6 January 2019
                11 February 2019
                March 2019
                11 August 2019
                : 2
                : 3
                : 228-235
                Affiliations
                [1 ]Department of Chemistry, Centre of Systems Biology and Human Health, State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Kowloon, Hong Kong
                [2 ]Department of Cellular and Molecular Medicine, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, United States
                [3 ]Department of Chemistry, New York University, New York, New York 10003 United States
                [4 ]Department of Chemistry, Sun Yat-Sen University, Guangzhou, China
                [5 ]Department of Chemistry, State Key Lab of Synthetic Chemistry, The University of Hong Kong, Hong Kong
                [6 ]NYU-ECNU Centre for Computational Chemistry at NYU Shanghai, Shanghai 200062, China
                [7 ]Computational Bioscience Research Centre (CBRC), CEMSE Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
                Author notes

                Author contributions

                J.X. and X.L prepared the proteins and performed the biochemical analyses. C.K.M.T., X.G., and S.W. performed aiQM/MM-MD simulations. H.Y.C. and X.L. performed Reverse phase-FPLC purification of thio-substituted oligonucleotides. C.K.M.T., J.X., P.P-H.C., F.K.S., D.W., Y.Z., and X.H. analysed the data. C.K.M.T., J.X., P.P-H.C., D.W., Y.Z., and X.H. wrote the manuscript with inputs from all authors. D.W., Y.Z. and X.H. directed and supervised the research.

                Article
                NIHMS1517152
                10.1038/s41929-019-0227-5
                6548511
                31179024

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