Hydrogen peroxide yields mechanistic insights into human mRNA capping enzyme function
| Autor: | Nicholas J. Mullen, David H. Price |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Guanylyltransferase Models Molecular lcsh:Medicine RNA polymerase II Biochemistry chemistry.chemical_compound Nucleic Acids Ribozymes Magnesium Recombinant Protein Purification Enzyme Inhibitors Hydrogen peroxide lcsh:Science Nucleoside-triphosphatase Multidisciplinary biology Messenger RNA Ribozyme Chemical Reactions Eukaryota Oxides Nucleoside-Triphosphatase Nucleotidyltransferases Enzymes Peroxides Chemistry Physical Sciences Research Article Chemical Elements RNA Caps Protein Purification Research and Analysis Methods 03 medical and health sciences Extraction techniques Protein Domains Capping enzyme Oxidation Humans Base Sequence lcsh:R Organisms Fungi Chemical Compounds Biology and Life Sciences Proteins Hydrogen Peroxide Yeast RNA extraction 030104 developmental biology chemistry Guanylyltransferase activity biology.protein Biocatalysis Enzymology RNA Triphosphatase lcsh:Q sense organs Purification Techniques |
| Zdroj: | PLoS ONE, Vol 12, Iss 10, p e0186423 (2017) PLoS ONE |
| ISSN: | 1932-6203 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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