The carboxyl terminal domain (CTD) of the large subunit of RNA Polymerase H is the target of multiple post-translational modifications, giving rise to two distinct in vivo isoforms designated RNA Pol IIA and RNA Pol IIO. Our lab has recently shown that the non-phosphorylated RNA Pol IIA isoform is extensively modified with an intracelfular form of protein glycosyiation called O-GIcNAc. The phosphorylated RNA Pol HO isoform contains no detectable levels of O-GlcNAc. This novel form of glycosyiation consists of single N-acetylglucosamine residues in O-glycosidic linkage to the side chain hydroxyls of serine and threonine on many resident nuclear and cytoplasmic proteins. To study the role of O-GIcNAc in RNA Pol n transcription, we carried out in vitro transcription in the presence of a specific inhibitor of the enzymatic removal of O-GlcNAc. We show here that inhibition of O-GlcNAc removal inhibits m vitro transcription in a dose dependent manner, suggesting that the regulated removal of O-GIcNAc is required during the transcription cycle. To further study the role of O-GlcNAc on RNA Pol H, we synthesized a glycopeptide containing one repeat of the CTD consensus sequence bearing a single O-GlcNAc residue. In vitro transcription in the presence of this glycopeptide significant inhibited transcription, while the unmodified CTD peptide exhibited only marginal inhibition, suggesting that the O-GlcNAc modification may mediate one or more critical interactions during the transcription process. These results suggest that the regulated addition and subsequent removal of O-GlcNAc may be required during each round of transcription and may function reciprocally with phosphorylation in the coordinate regulation of RNA Polymerase 0. Current efforts are aimed at determining the glycosyiation status of the CTD as well as other proteins during transcription initiation and elongation.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology