Ubiquitination of RNA polymerase II large subunit signaled by phosphorylation of carboxyl-terminal domain

Ubiquitination of RNA polymerase II large subunit signaled by phosphorylation of carboxyl-terminal domain

Akira Mitsui* and
Phillip A. Sharp†

Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307

Contributed by Phillip A. Sharp

Abstract

A sensitive assay using biotinylated ubiquitin revealed extensive ubiquitination of the large subunit of RNA polymerase II during incubations of transcription reactions in vitro. Phosphorylation of the repetitive carboxyl-terminal domain of the large subunit was a signal for ubiquitination. Specific inhibitors of cyclin-dependent kinase (cdk)-type kinases suppress the ubiquitination reaction. These kinases are components of transcription factors and have been shown to phosphorylate the carboxyl-terminal domain. In both regulation of transcription and DNA repair, phosphorylation of the repetitive carboxyl-terminal domain by kinases might signal degradation of the polymerase.

Footnotes

↵ * Present address: Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki, Japan.
↵ † To whom reprint requests should be addressed. e-mail: sharppa{at}mit.edu.
ABBREVIATIONS:

Pol II,

RNA polymerase II;

Pol II LS,

large subunit of Pol II;

CS-A and CS-B,

Cockayne syndrome complementation groups A and B;

CTD,

carboxyl-terminal domain;

cdk,

cyclin-dependent kinase;

DRB,

5,6-dichloro-1-β-d-ribofuranosylbenzimidazole;

H7,

1-(5-isoquinolinylsulfonyl)-2-methylpiperazine;

H8,

N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide;

HRP,

horseradish peroxidase;

GST,

glutathione S-transferase;

bio-Ub,

biotinylated ubiquitin