title: Transcriptionally active TFIIH of the early-diverged eukaryote Trypanosoma brucei harbors two novel core subunits but not a cyclin-activating kinase complex.
abstract: Trypanosoma brucei is a member of the early-diverged, protistan family Trypanosomatidae and a lethal parasite causing African Sleeping Sickness in humans. Recent studies revealed that T. brucei harbors extremely divergent orthologues of the general transcription factors TBP, TFIIA, TFIIB and TFIIH and showed that these factors are essential for initiating RNA polymerase II-mediated synthesis of spliced leader (SL) RNA, a trans splicing substrate and key molecule in trypanosome mRNA maturation. In yeast and metazoans, TFIIH is composed of a core of seven conserved subunits and the ternary cyclin-activating kinase (CAK) complex. Conversely, only four TFIIH subunits have been identified in T. brucei. Here, we characterize the first protistan TFIIH which was purified in its transcriptionally active form from T. brucei extracts. The complex consisted of all seven core subunits but lacked the CAK sub-complex; instead it contained two trypanosomatid-specific subunits, which were indispensable for parasite viability and SL RNA gene transcription. These findings were corroborated by comparing the molecular structures of trypanosome and human TFIIH. While the ring-shaped core domain was surprisingly congruent between the two structures, trypanosome TFIIH lacked the knob-like CAK moiety and exhibited extra densities on either side of the ring, presumably due to the specific subunits.
authors.affiliation: Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3301, USA.
authors.forename: Ju Huck
authors.initials: JH
authors.lastname: Lee
authors.forename: Hyun Suk
authors.initials: HS
authors.lastname: Jung
authors.forename: Arthur
authors.initials: A
authors.lastname: Günzl
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D018844:Cyclin-Dependent Kinases / Q000032:analysis; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D015800:Protozoan Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D034622:RNA Interference; D020038:RNA, Spliced Leader / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D051758:Transcription Factor TFIIH / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*
chemical_list: D021122:Protein Subunits; D015800:Protozoan Proteins; D020038:RNA, Spliced Leader; D051758:Transcription Factor TFIIH; D018844:Cyclin-Dependent Kinases