Affiliation:
1. Department of Genetics and Developmental Biology and Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030-3710
Abstract
ABSTRACT
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.
Publisher
American Society for Microbiology
Subject
Cell Biology,Molecular Biology
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