Journal article
Regulation of RNA Polymerase II Termination by Phosphorylation of Gdown1
The Journal of biological chemistry, Vol.289(18), pp.12657-12665
05/02/2014
DOI: 10.1074/jbc.M113.537662
PMCID: PMC4007455
PMID: 24634214
Abstract
Background:
Gdown1 is a substoichiometric subunit of RNA polymerase II that blocks termination by TTF2 and the function of TFIIF during initiation and elongation.
Results:
Domains of Gdown1 responsible for inhibiting TTF2 and TFIIF as well as a controlling phosphorylation site are identified.
Conclusion:
Gdown1 can be regulated by phosphorylation of Ser-270.
Significance:
Our results reveal a mechanism that allows termination of all polymerases during mitosis.
Gdown1 is a substoichiometric subunit of RNA polymerase II (Pol II) that has been recently demonstrated to be involved in stabilizing promoter-proximal paused Pol II. It was shown to inhibit termination of Pol II by transcription termination factor 2 (TTF2) as well as block elongation stimulation by transcription factor IIF (TFIIF). Here, using
in vitro
transcription assays, we identified two functional domains in Gdown1. Although both are required to maintain a tight association with Pol II, the N- and C-terminal domains are responsible for blocking TTF2 and TFIIF, respectively. A highly conserved LPDKG motif found in the N-terminal domain of Gdown1 is also highly conserved in TTF2. Deletion of this motif eliminated the TTF2 inhibitory activity of Gdown1. We identified a phosphorylated form of Gdown1 with altered mobility in SDS-PAGE that appears during mitosis. A kinase in HeLa nuclear extract that caused the shift was partially purified.
In vitro
, Gdown1 phosphorylated by this kinase demonstrated reduced activity in blocking both TTF2 and TFIIF because of its reduced affinity for Pol II. Mass spectrometry identified Ser-270 as the site of this phosphorylation. An S270A mutation was not phosphorylated by the partially purified kinase, and an S270E mutation partially mimicked the properties of phospho-Gdown1. Gdown1 Ser-270 phosphorylation occurs predominately during mitosis, and we suggest that this would enable TTF2 to terminate all Pol II even if it is associated with Gdown1.
Details
- Title: Subtitle
- Regulation of RNA Polymerase II Termination by Phosphorylation of Gdown1
- Creators
- Jiannan Guo - From the Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242Michael E Turek - From the Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242David H Price - From the Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242
- Resource Type
- Journal article
- Publication Details
- The Journal of biological chemistry, Vol.289(18), pp.12657-12665
- DOI
- 10.1074/jbc.M113.537662
- PMID
- 24634214
- PMCID
- PMC4007455
- NLM abbreviation
- J Biol Chem
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- Publisher
- American Society for Biochemistry and Molecular Biology; 9650 Rockville Pike, Bethesda, MD 20814, U.S.A
- Grant note
- GM35500 / National Institutes of Health
- Alternative title
- Regulation of Gdown1 by Phosphorylation
- Language
- English
- Date published
- 05/02/2014
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9984024404402771
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