Characterization of a multisubunit transcription factor complex essential for spliced-leader RNA gene transcription in Trypanosoma brucei.
In the unicellular human parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., the spliced-leader (SL) RNA is a key molecule in gene expression donating its 5'-terminal region in SL addition trans splicing of nuclear pre-mRNA. While there is no evidence that this process exists in mammals, it is obligatory in mRNA maturation of trypanosomatid parasites. Hence, throughout their life cycle, these organisms crucially depend on high levels of SL RNA synthesis. As putative SL RNA gene transcription factors, a partially characterized small nuclear RNA-activating protein complex (SNAP(c)) and the TATA-binding protein related factor 4 (TRF4) have been identified thus far. Here, by tagging TRF4 with a novel epitope combination termed PTP, we tandem affinity purified from crude T. brucei extracts a stable and transcriptionally active complex of six proteins. Besides TRF4 these were identified as extremely divergent subunits of SNAP(c) and of transcription factor IIA (TFIIA). The latter finding was unexpected since genome databases of trypanosomatid parasites appeared to lack general class II transcription factors. As we demonstrate, the TRF4/SNAP(c)/TFIIA complex binds specifically to the SL RNA gene promoter upstream sequence element and is absolutely essential for SL RNA gene transcription in vitro.
Schimanski
Bernd
B
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, 06030-3710, USA.
Nguyen
Tu N
TN
Günzl
Arthur
A
D020121:5' Untranslated Regions; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004330:Drosophila; D000939:Epitopes / Q000737:chemistry; D016678:Genome; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012326:RNA Splicing; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000494:pharmacology; D035181:TATA-Box Binding Protein / Q000378:metabolism; D035561:Transcription Factor TFIIA / Q000737:chemistry; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei
D020121:5' Untranslated Regions; D002868:Chromosomal Proteins, Non-Histone; D000939:Epitopes; D009687:Nuclear Proteins; D010455:Peptides; C106703:SNAPC protein complex, human; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D014157:Transcription Factors; D013395:Sucrose; D012313:RNA; D004259:DNA-Directed DNA Polymerase; C118146:TENT4A protein, human