RNA polymerase II is a glycoprotein. Modification of the COOH-terminal domain by O-GlcNAc

W.G Kelly,M.E Dahmus,G.W Hart

doi:10.1016/s0021-9258(18)82216-5

Abstract

The largest subunit of mammalian RNA polymerase II (RNAP II) contains at its carboxyl terminus an unusual domain consisting of 52 tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain, designated the COOH-terminal domain (CTD), is essential for viability and is extensively phosphorylated during the transition from preinitiation complex assembly to elongation (1). Indeed, phosphorylation of the CTD may play an important regulatory role in this transition. We show here that the CTD is also modified by a novel form of protein glycosylation, O-GlcNAc. This modification has been found on numerous transcription factors and other nuclear and cytosolic proteins (2). Glycopeptides obtained by proteolytic digestion of the CTD were purified by reverse-phase high performance liquid chromatography and sequenced. Results from such experiments suggest that glycosylation occurs at multiple sites throughout the CTD, similar to the phosphorylation of this domain. The carbohydrate, however, is not detectable on the phosphorylated form of the enzyme. This observation is consistent with the idea that phosphorylation and glycosylation are mutually exclusive modifications. The CTD of RNAP II, therefore, appears to exist in three distinct conformational states: unmodified, phosphorylated, and glycosylated. The differential modification of the CTD may play an important role in the regulated expression of genes transcribed by RNA polymerase II.

Highlights

From the Department ofBiological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205 and the $Department of Biochemistry and Biophysics, University ofCalifornia,Davis, California95616
We show here that the CTD is modified by a novel form of protein glycosylation, 0-GlcNAcT. his modification has been found on numerous transcription factors and other established that this domain is essential for viability [6,7,8,9]
Change in glycosylation patterns has been observed in nuclear proteins during the different stagesof the cell cycle, with a pronounced increase in nuclear glycosylation seen at the Gl/S boundary, followed by a decrease atmito ~ i sR. ~ecently it has been shown that the 0-GlcNAcs on cyto

Summary

RESULTS

14 a milk galactosyltransferase (GTase) can catalyze the transfer of [3H]galactose from the nucleotide sugar donor UDP-[3H] FIG. 2. The peptide sequences obtained could be the carbohydrate is spread throughout this domain This is from contaminating peptides thathave the same RP-HPLC illustrated in Fig. 7B inwhich all of the sequencesin the mobilities as theglycosylated species,since thelow abundance mouse CTD thatmay be accounted fobr y these glycopeptides radioactive peptides would be virtually undetectable by gas- are in bold type and underlined. It has been our experienceh,owever, based The exact site of 0-GlcNAc attachment was identified on onin uitro, andnaturally glycosylated peptides,thatthe three of these glycopeptides: the peptides S/T-P-S/T-S-P, Taddition of carbohydrate to peptides similar in sequence to P-T-S-P-N, and S-P-T-S-P-T(Fig. 8) These were obtained those described below does not detectably affect their retention times in the RP-HPLC systemwe use (data not shown).

PRONASEGLYCOPEPTIDES

DISCUSSION