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    Intrinsic cleavage of RNA polymerase II adopts a nucleobase-independent mechanism assisted by transcript phosphate

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    Type
    Article
    Authors
    Tse, Carmen Ka Man
    Xu, Jun
    Xu, Liang
    Sheong, Fu Kit
    Wang, Shenglong
    Chow, Hoi Yee
    Gao, Xin cc
    Li, Xuechen
    Cheung, Peter Pak-Hang
    Wang, Dong
    Zhang, Yingkai
    Huang, Xuhui
    KAUST Department
    Computational Bioscience Research Center (CBRC)
    Computer Science Program
    Computer, Electrical and Mathematical Sciences and Engineering (CEMSE) Division
    Structural and Functional Bioinformatics Group
    KAUST Grant Number
    OSR-2016-CRG5-3007
    Date
    2019-02-11
    Online Publication Date
    2019-02-11
    Print Publication Date
    2019-03
    Permanent link to this record
    http://hdl.handle.net/10754/631390
    
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    Abstract
    RNA 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.
    Citation
    Tse 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.
    Sponsors
    We 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.
    Publisher
    Springer Nature
    Journal
    Nature Catalysis
    DOI
    10.1038/s41929-019-0227-5
    Additional Links
    https://www.nature.com/articles/s41929-019-0227-5
    ae974a485f413a2113503eed53cd6c53
    10.1038/s41929-019-0227-5
    Scopus Count
    Collections
    Articles; Structural and Functional Bioinformatics Group; Computer Science Program; Computational Bioscience Research Center (CBRC); Computer, Electrical and Mathematical Science and Engineering (CEMSE) Division

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