Regulation of RNA Polymerase II Termination by Phosphorylation of Gdown1 [RNA]

March 14th, 2014 by Guo, J., Turek, M. E., Price, D. H.

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 TTF2 as well as block elongation stimulation by TFIIF. Here, using in vitro transcription assays, we identified two functional domains in Gdown1. While 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 due to its reduced affinity for Pol II. Mass spectrometry identified S270 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 S270 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.