TRYPANOSOMA-BRUCEI RNA-POLYMERASE-II IS PHOSPHORYLATED IN THE ABSENCEOF CARBOXYL-TERMINAL DOMAIN HEPTAPEPTIDE REPEATS

Formation of an RNA polymerase II transcription initiation complex req uires binding of a polymerase that contains a non-phosphorylated large st subunit carboxyl-terminal domain (CTD). Polymerase binding is follo wed by elongation after phosphorylation of the CTD by a CTD kinase. Ph osphorylation sites are within the repeating heptapeptide motifs which characterize the CTD of all eukaryotic RNA polymerase IIs. In contras t to all other eukaryotes studied, the trypanosome genome contains two genetic loci which encode the large subunit of RNA polymerase II; bot h genes lack CTD heptapeptide repeat structures. We have examined whet her Trypanosoma brucei RNA polymerase II, despite its unique CTD domai n, is phosphorylated when isolated from elongating transcription compl exes. Elongating trypanosome RNA polymerases were photoaffinity labele d during nuclear run-on assays. The identity of the labeled proteins w as established by immunoblotting and immunoprecipitation using polymer ase-specific antisera, Analysis of the largest subunit of RNA polymera se II revealed the expected 195-kDa species and an additional larger 2 20-kDa species. The apparent molecular weight of this larger form of R NA polymerase II decreased incrementally as a function of incubation w ith increasing concentrations of calf intestinal phosphatase. These re sults show that extensive phosphorylation of the largest subunit of RN A polymerase-II is a conserved feature between trypanosomes and higher eukaryotes despite the absence of a typical CTD domain.