One- and two-hybrid analysis of the interactions between components of the Trypanosoma cruzi spliced leader RNA gene promoter binding complex

Abstract

The spliced leader (SL) RNA gene promoter is the only RNA polymerase II-dependent promoter characterized to date in trypanosomatids. Transcription of this small nuclear RNA is critical for trypanosomatid cell life because it is needed for polycistronic primary transcripts processing into individual translatable mRNAs. In recent years, a set of divergent fundamental transcription factors required for SL RNA gene transcription have been identified in different trypanosomatids. By means of a yeast two-hybrid system, we analyzed the protein–protein interactions between components of the SL RNA gene promoter binding complex. We also studied the interactions of already described motifs of TATA-binding protein (TBP) and transcription factor II B (TFIIB) orthologs separately. This was followed by investigations of DNA-protein interactions within the SL RNA gene promoter binding complex using one-hybrid analysis. Our results suggest that the complex has two “cores” which contact the promoter DNA, trypanosomal small nuclear RNA activating protein complex (tSNAPc), which has strong interactions between its subunits and a more labile TBP-TFIIA sub-complex.