Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue

Lin-Tai Da; Fátima Pardo-Avila; Liang Xu; Daniel-Adriano Silva; Lu Zhang; Xin Gao; Dong Wang; Xuhui Huang

doi:10.1038/ncomms11244

Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue

Lin-Tai Da, Fátima Pardo-Avila, Liang Xu, Daniel-Adriano Silva, Lu Zhang, Xin Gao, Dong Wang, Xuhui Huang

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71 Scopus citations

Abstract

The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.

Original language	English (US)
Journal	Nature Communications
Volume	7
Issue number	1
DOIs	https://doi.org/10.1038/ncomms11244
State	Published - Apr 19 2016

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: X.H. acknowledges the Hong Kong Research Grants Council (16302214, 609813, HKUST C6009-15G, AoE/M-09/12, T13-607/12R, and M-HKUST601/13) and National Science Foundation of China 21273188. D.W. acknowledges the NIH (GM102362), Kimmel Scholars award from the Sidney Kimmel Foundation for Cancer Research, start-up funds from Skaggs School of Pharmacy and Pharmaceutical Sciences, UCSD and Academic Senate Research Award from UCSD. X.G. was supported by funding from the King Abdullah University of Science and Technology. F.P. acknowledges the support from Hong Kong PhD Fellowship Scheme (2011/12) and the partial support for PhD studies from the CONACYT. This research made use of the resources of the Supercomputing Laboratory at King Abdullah University of Science and Technology. We thank Dr Jeffery Strathern (NCI) for providing yeast strain containing Pol II Rpb1 T831A mutant and Dr Mikhail Kashlev for providing purified Pol II Rpb1 T831A mutant.

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https://repository.kaust.edu.sa/bitstream/10754/606053/1/ncomms11244.pdf

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title = "Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue",

abstract = "The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.",

author = "Lin-Tai Da and F{\'a}tima Pardo-Avila and Liang Xu and Daniel-Adriano Silva and Lu Zhang and Xin Gao and Dong Wang and Xuhui Huang",

note = "KAUST Repository Item: Exported on 2020-10-01 Acknowledgements: X.H. acknowledges the Hong Kong Research Grants Council (16302214, 609813, HKUST C6009-15G, AoE/M-09/12, T13-607/12R, and M-HKUST601/13) and National Science Foundation of China 21273188. D.W. acknowledges the NIH (GM102362), Kimmel Scholars award from the Sidney Kimmel Foundation for Cancer Research, start-up funds from Skaggs School of Pharmacy and Pharmaceutical Sciences, UCSD and Academic Senate Research Award from UCSD. X.G. was supported by funding from the King Abdullah University of Science and Technology. F.P. acknowledges the support from Hong Kong PhD Fellowship Scheme (2011/12) and the partial support for PhD studies from the CONACYT. This research made use of the resources of the Supercomputing Laboratory at King Abdullah University of Science and Technology. We thank Dr Jeffery Strathern (NCI) for providing yeast strain containing Pol II Rpb1 T831A mutant and Dr Mikhail Kashlev for providing purified Pol II Rpb1 T831A mutant.",

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AU - Da, Lin-Tai

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AU - Zhang, Lu

AU - Gao, Xin

AU - Wang, Dong

AU - Huang, Xuhui

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N2 - The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.

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Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue

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