Hydrogen peroxide yields mechanistic insights into human mRNA capping enzyme function

Nicholas J Mullen; David H Price

doi:10.1371/journal.pone.0186423

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Hydrogen peroxide yields mechanistic insights into human mRNA capping enzyme function

Journal article

Open access

Peer reviewed

Hydrogen peroxide yields mechanistic insights into human mRNA capping enzyme function

Nicholas J Mullen and David H Price

PloS one, Vol.12(10), pp.e0186423-e0186423

2017

DOI: 10.1371/journal.pone.0186423

PMCID: PMC5640233

PMID: 29028835

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Abstract

Capping of nascent RNA polymerase II (Pol II) transcripts is required for gene expression and the first two steps are catalyzed by separate 5' triphosphatase and guanylyltransferase activities of the human capping enzyme (HCE). The cap is added co-transcriptionally, but how the two activities are coordinated is unclear. Our previous in vitro work has suggested that an unidentified factor modulates the minimum length at which nascent transcripts can be capped. Using the same well-established in vitro system with hydrogen peroxide as a capping inhibitor, we show that this unidentified factor targets the guanylyltransferase activity of HCE. We also uncover the mechanism of HCE inhibition by hydrogen peroxide, and by using mass spectrometry demonstrate that the active site cysteine residue of the HCE triphosphatase domain becomes oxidized. Using recombinant proteins for the two separated HCE domains, we provide evidence that the triphosphatase normally acts on transcripts shorter than can be acted upon by the guanylyltransferase. Our further characterization of the capping reaction dependence on transcript length and its interaction with the unidentified modulator of capping raises the interesting possibility that the capping reaction could be regulated.

Nucleotidyltransferases - chemistry

Biocatalysis

Humans

Enzyme Inhibitors - pharmacology

Hydrogen Peroxide - pharmacology

Models, Molecular

RNA Caps - metabolism

Nucleoside-Triphosphatase - antagonists & inhibitors

Nucleoside-Triphosphatase - metabolism

Base Sequence

Protein Domains

Nucleotidyltransferases - antagonists & inhibitors

Nucleotidyltransferases - metabolism

RNA Caps - genetics

Nucleoside-Triphosphatase - chemistry

Details

Title: Subtitle: Hydrogen peroxide yields mechanistic insights into human mRNA capping enzyme function
Creators: Nicholas J Mullen - Department of Biochemistry, University of Iowa, Iowa City, Iowa, United States of America
David H Price - Department of Biochemistry, University of Iowa, Iowa City, Iowa, United States of America
Resource Type: Journal article
Publication Details: PloS one, Vol.12(10), pp.e0186423-e0186423
DOI: 10.1371/journal.pone.0186423
PMID: 29028835
PMCID: PMC5640233
NLM abbreviation: PLoS One
ISSN: 1932-6203
eISSN: 1932-6203
Publisher: Public Library of Science; United States
Grant note: R01 GM113935 / NIGMS NIH HHS R01 GM035500 / NIGMS NIH HHS
Language: English
Date published: 2017
Academic Unit: Biochemistry and Molecular Biology
Record Identifier: 9984024552002771

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