Abstract

RNA polymerase II (Pol II) surveils the genome, pausing as it encounters DNA lesions and base modifications and initiating signals for DNA repair among other important regulatory events. Recent work suggests that Pol II pauses at 5-carboxycytosine (5caC), an epigenetic modification of cytosine, because of a specific hydrogen bond between the carboxyl group of 5caC and a specific residue in fork loop 3 of Pol II. This hydrogen bond compromises productive NTP binding and slows down elongation. Apart from this specific interaction, the carboxyl group of 5caC can potentially interact with numerous charged residues in the cleft of Pol II. However, it is not clear how other interactions between Pol II and 5caC contribute to pausing. In this study, we use Markov state models (a type of kinetic network models) built from extensive molecular dynamics simulations to comprehensively study the impact of 5caC on Pol II translocation. We describe two translocation intermediates with specific interactions that prevent the template base from loading into the Pol II active site. In addition to the previously observed state with 5caC constrained by fork loop 3, we discovered a new intermediate state with a hydrogen bond between 5caC and fork loop 2. Surprisingly, we find that 5caC may curb translocation by suppressing kinking of the helix bordering the active site (the bridge helix) because its high flexibility is critical to translocation. Our work provides new insights into how epigenetic modifications of genomic DNA can modulate Pol II translocation, inducing pauses in transcription.

Highlights

  • Epigenetic modifications of the genomic DNA are part of a vast suite of regulatory machinery in higher eukaryotes

  • In a structure of Pol RNA polymerase II (II) elongation complex with 5caC (PDB ID: 4Y52), the electron density of the 5caC suggests that two states are present: a canonical post-translocation state and an intermediate state in which the base is held by a hydrogen bond with Q531 in a midway position between the +1 and +2 template sites

  • We studied polymerase II (Pol II) translocation using Markov state model (MSM) based on molecular dynamics (MD) simulations to resolve the intermediate states that pause transcription with 5caC

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Summary

Introduction

Epigenetic modifications of the genomic DNA are part of a vast suite of regulatory machinery in higher eukaryotes. We studied Pol II translocation using MSMs based on MD simulations to resolve the intermediate states that pause transcription with 5caC.

Results
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