Nucleotide Loading Modes of Human RNA Polymerase II as Deciphered by Molecular Simulations.

Nicolas E J Génin,Robert O J Weinzierl

doi:10.3390/biom10091289

Nicolas E J Génin, Robert O J Weinzierl

Open Access

https://doi.org/10.3390/biom10091289

Copy DOI

Journal: Biomolecules	Publication Date: Sep 7, 2020
Citations: 2	License type: CC BY 4.0

Affiliation: University of Orléans, Imperial College London

Abstract

Mapping the route of nucleoside triphosphate (NTP) entry into the sequestered active site of RNA polymerase (RNAP) has major implications for elucidating the complete nucleotide addition cycle. Constituting a dichotomy that remains to be resolved, two alternatives, direct NTP delivery via the secondary channel (CH2) or selection to downstream sites in the main channel (CH1) prior to catalysis, have been proposed. In this study, accelerated molecular dynamics simulations of freely diffusing NTPs about RNAPII were applied to refine the CH2 model and uncover atomic details on the CH1 model that previously lacked a persuasive structural framework to illustrate its mechanism of action. Diffusion and binding of NTPs to downstream DNA, and the transfer of a preselected NTP to the active site, are simulated for the first time. All-atom simulations further support that CH1 loading is transcription factor IIF (TFIIF) dependent and impacts catalytic isomerization. Altogether, the alternative nucleotide loading systems may allow distinct transcriptional landscapes to be expressed.

Highlights

RNA polymerases (RNAPs) are nanoscopic machines responsible for transcribing sections of the information stored in DNA into RNA
The initial system was the 10-subunit human RNAPII, extracted from PDB#5IY9 [44], with the transcription factor IIF (TFIIF) subunit RAP74 extended to amino acid position 227 [45], the RNA chain extended to i − 16, and the double-stranded DNA ranged from i − 30 to i + 20
The conjunction of the upstream strain induced by TFIIF Modules 2 to 4 on one side, with the downstream anchor provided by Module 1 on the other side, resulted in folding of non-template DNA (ntDNA) in between these modules (Figure S2C,D)

Summary

Introduction

RNA polymerases (RNAPs) are nanoscopic machines responsible for transcribing sections of the information stored in DNA into RNA. Experimental data support that the pathway can accommodate substrate binding to downstream DNA at the i + 2, i + 3, or even up to the i + 4 registers, acting as secondary templated sites, before NTPs are incrementally shifted to the catalytic site. This concept was based on compelling evidence comparing the fate of i + 1 (or subsequent i + 2) nucleotide with and without preincubation of the templated nucleotides [15,16,17,18,19]. Based on persuasive functional data, it has been challenging to reconcile the CH1 hypothesis with the available structural material, as tight protein electrostatic contacts with the downstream DNA helix did not appear to leave room for NTPs to enter alongside

Methods

Results

Discussion

Conclusion