title: The second largest subunit of Trypanosoma brucei's multifunctional RNA polymerase I has a unique N-terminal extension domain.
abstract: In the protist parasite Trypanosoma brucei, RNA polymerase (pol) I transcribes the large ribosomal RNA gene unit and, in addition, variant surface glycoprotein gene expression sites and procyclin gene transcription units. The multifunctional role of RNA pol I in this organism is unique among eukaryotes, but only its largest subunit TbRPA1 has been characterized thus far. We have recently established the procyclic cell line RPIC which exclusively expresses RNA pol I tagged with the protein C epitope at the TbRPA1 C-terminus. In the present study, we prepared RPIC cell extracts and immunopurified RNA pol I using anti-protein C affinity matrix under high stringency conditions. We were able to identify five specific polypeptides on a silver-stained polyacrylamide-SDS gel with apparent molecular weights of 200, 180, 55, 29, and 22 kDa. Interestingly, the second largest subunit, TbRPA2, is 42-58 kDa larger than counterparts of other organisms. We have cloned and sequenced the complete TbRPA2 cDNA and found an open reading frame for a polypeptide of 179.5 kDa. The deduced amino acid sequence of TbRPA2 contains a unique N-terminal domain of approximately 250 amino acids. By raising a polyclonal antibody against a N-terminal peptide sequence of TbRPA2, we could specifically detect this polypeptide in immunoblots showing that it co-purifies with epitope-tagged TbRPA1. Moreover, we identified the homologous gene sequence LmRPA2 in Leishmania major and found that it encodes a homologous extension domain. Therefore, the N-terminal extra domain in trypanosomatid RPA2 polypeptides may serve a parasite-specific function.
authors.affiliation: Medizinisch-Naturwissenschaftliches Forschungszentrum, Ob dem Himmelreich 7, 72074 Tübingen, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Schimanski
authors.forename: Birgit
authors.initials: B
authors.lastname: Klumpp
authors.forename: Gabriele
authors.initials: G
authors.lastname: Laufer
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Marhöfer
authors.forename: Paul M
authors.initials: PM
authors.lastname: Selzer
authors.forename: Arthur
authors.initials: A
authors.lastname: Günzl
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D051379:Mice; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D012318:RNA Polymerase I / Q000737:chemistry* / Q000235:genetics; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
chemical_list: D021122:Protein Subunits; D016053:RNA, Protozoan; D012335:RNA, Ribosomal; D011994:Recombinant Proteins; D012319:RNA Polymerase II; D012318:RNA Polymerase I