Characterization of a TFIIH homologue from Trypanosoma brucei.
Trypanosomes are protozoans showing unique transcription characteristics. We describe in Trypanosoma brucei a complex homologous to TFIIH, a multisubunit transcription factor involved in the control of transcription by RNA Pol I and RNA Pol II, but also in DNA repair and cell cycle control. Bioinformatics analyses allowed the detection of five genes encoding four putative core TFIIH subunits (TbXPD, TbXPB, Tbp44, Tbp52), including a novel XPB variant, TbXPBz. In all cases sequences known to be important for TFIIH functions were conserved. We performed a molecular analysis of this core complex, focusing on the two subunits endowed with a known enzymatic (helicase) activity, XPD and XPB. The involvement of these T. brucei proteins in a bona fide TFIIH core complex was supported by (i) colocalization by immunofluorescence in the nucleus, (ii) direct physical interaction of TbXPD and its interacting regulatory subunit Tbp44 as determined by double-hybrid assay and tandem affinity purification of the core TFIIH, (iii) involvement of the core proteins in a high molecular weight complex and (iv) occurrence of transcription, cell cycle and DNA repair phenotypes upon either RNAi knock-down or overexpression of essential subunits.
Lecordier
Laurence
L
Laboratory of Molecular Parasitology, Institute of Molecular Biology and Medicine, Université Libre de Bruxelles, 12, rue des Professeurs Jeener et Brachet, B-6041 Gosselies, Belgium.
Devaux
Sara
S
Uzureau
Pierrick
P
Dierick
Jean François
JF
Walgraffe
David
D
Poelvoorde
Philippe
P
Pays
Etienne
E
Vanhamme
Luc
L
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence; D004265:DNA Helicases; D004260:DNA Repair; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid; D051758:Transcription Factor TFIIH / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000235:genetics*; D020798:Two-Hybrid System Techniques
D021122:Protein Subunits; D051758:Transcription Factor TFIIH; D004265:DNA Helicases