Regulation of RNA polymerase II termination by phosphorylation of Gdown1

J Biol Chem. 2014 May 2;289(18):12657-65. doi: 10.1074/jbc.M113.537662. Epub 2014 Mar 14.

Abstract

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.

Keywords: Gdown1; Mitosis; Phosphorylation; RNA Polymerase II; TFIIF; TTF2; Transcription Elongation Factors; Transcription Termination.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism*
  • Amino Acid Motifs / genetics
  • Amino Acid Sequence
  • Binding Sites / genetics
  • Binding, Competitive
  • Blotting, Western
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • HeLa Cells
  • Humans
  • Mass Spectrometry
  • Mitosis / genetics
  • Molecular Sequence Data
  • Mutation
  • Phosphorylation
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA Polymerase II / chemistry
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • Sequence Homology, Amino Acid
  • Serine / chemistry
  • Serine / genetics
  • Serine / metabolism*
  • Transcription Factors / chemistry
  • Transcription Factors / metabolism*
  • Transcription Factors, TFII / chemistry
  • Transcription Factors, TFII / metabolism
  • Transcription Termination, Genetic*

Substances

  • DNA-Binding Proteins
  • POLR2M protein, human
  • Transcription Factors
  • Transcription Factors, TFII
  • Serine
  • RNA Polymerase II
  • Adenosine Triphosphatases
  • TTF2 protein, human
  • transcription factor TFIIF