Trypanosoma brucei RNA polymerase II is phosphorylated in the absence of carboxyl-terminal domain heptapeptide repeats.
Formation of an RNA polymerase II transcription initiation complex requires binding of a polymerase that contains a non-phosphorylated largest subunit carboxyl-terminal domain (CTD). Polymerase binding is followed by elongation after phosphorylation of the CTD by a CTD kinase. Phosphorylation sites are within the repeating heptapeptide motifs which characterize the CTD of all eukaryotic RNA polymerase IIs. In contrast to all other eukaryotes studied, the trypanosome genome contains two genetic loci which encode the large subunit of RNA polymerase II; both genes lack CTD heptapeptide repeat structures. We have examined whether Trypanosoma brucei RNA polymerase II, despite its unique CTD domain, is phosphorylated when isolated from elongating transcription complexes. Elongating trypanosome RNA polymerases were photoaffinity labeled during nuclear run-on assays. The identity of the labeled proteins was established by immunoblotting and immunoprecipitation using polymerase-specific antisera. Analysis of the largest subunit of RNA polymerase II revealed the expected 195-kDa species and an additional larger 220-kDa species. The apparent molecular weight of this larger form of RNA polymerase II decreased incrementally as a function of incubation with increasing concentrations of calf intestinal phosphatase. These results show that extensive phosphorylation of the largest subunit of RNA polymerase-II is a conserved feature between trypanosomes and higher eukaryotes despite the absence of a typical CTD domain.
Chapman
A B
AB
Intercampus Program in Molecular Parasitology, University of California, Berkeley.
Agabian
N
N
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D017173:Caenorhabditis elegans / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology*; D016384:Consensus Sequence; D004330:Drosophila / Q000201:enzymology; D005982:Glutathione Transferase / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010766:Phosphorylation; D010961:Plasmodium / Q000201:enzymology; D012318:RNA Polymerase I / Q000302:isolation & purification; D012319:RNA Polymerase II / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism
D000345:Affinity Labels; D011993:Recombinant Fusion Proteins; C059149:4-thiouridine triphosphate; D005982:Glutathione Transferase; D012319:RNA Polymerase II; D012318:RNA Polymerase I; D014544:Uridine Triphosphate