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

Carmen Ka Man Tse; Jun Xu; Liang Xu; Fu Kit Sheong; Shenglong Wang; Hoi Yee Chow; Xin Gao; Xuechen Li; Peter Pak-Hang Cheung; Dong Wang; Yingkai Zhang; Xuhui Huang

doi:10.1038/s41929-019-0227-5

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

Carmen Ka Man Tse, Jun Xu, Liang Xu, Fu Kit Sheong, Shenglong Wang, Hoi Yee Chow, Xin Gao, Xuechen Li, Peter Pak-Hang Cheung, Dong Wang, Yingkai Zhang, Xuhui Huang

Research output: Contribution to journal › Article › peer-review

11 Scopus citations

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.

Original language	English (US)
Pages (from-to)	228-235
Number of pages	8
Journal	Nature Catalysis
Volume	2
Issue number	3
DOIs	https://doi.org/10.1038/s41929-019-0227-5
State	Published - Feb 11 2019

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledged KAUST grant number(s): OSR-2016-CRG5-3007
Acknowledgements: 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.

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10.1038/s41929-019-0227-5

https://repository.kaust.edu.sa/bitstream/10754/631390/1/769_2_merged_1543920740.pdf

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@article{f0d15ce3166d43cb9674e4e7c3e9e4bc,

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

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.",

author = "Tse, {Carmen Ka Man} and Jun Xu and Liang Xu and Sheong, {Fu Kit} and Shenglong Wang and Chow, {Hoi Yee} and Xin Gao and Xuechen Li and Cheung, {Peter Pak-Hang} and Dong Wang and Yingkai Zhang and Xuhui Huang",

note = "KAUST Repository Item: Exported on 2020-10-01 Acknowledged KAUST grant number(s): OSR-2016-CRG5-3007 Acknowledgements: 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.",

year = "2019",

month = feb,

day = "11",

doi = "10.1038/s41929-019-0227-5",

language = "English (US)",

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T1 - Intrinsic cleavage of RNA polymerase II adopts a nucleobase-independent mechanism assisted by transcript phosphate

AU - Tse, Carmen Ka Man

AU - Xu, Jun

AU - Xu, Liang

AU - Sheong, Fu Kit

AU - Wang, Shenglong

AU - Chow, Hoi Yee

AU - Gao, Xin

AU - Li, Xuechen

AU - Cheung, Peter Pak-Hang

AU - Wang, Dong

AU - Zhang, Yingkai

AU - Huang, Xuhui

N1 - KAUST Repository Item: Exported on 2020-10-01 Acknowledged KAUST grant number(s): OSR-2016-CRG5-3007 Acknowledgements: 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.

PY - 2019/2/11

Y1 - 2019/2/11

N2 - 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.

AB - 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.

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U2 - 10.1038/s41929-019-0227-5

DO - 10.1038/s41929-019-0227-5

M3 - Article

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SP - 228

EP - 235

JO - Nature Catalysis

JF - Nature Catalysis

IS - 3

ER -

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

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