The trypanosomatid parasites Trypanosoma brucei, T. cruzi and Leishmania spp. (Tritryp) cause the major human diseases African Sleeping Sickness, Chagas'disease, and Leishmaniasis, respectively. Drugs for these diseases are few and toxic and parasite resistance to these drugs is on the rise. Thus, it becomes increasingly important to find new anti-parasitic targets and develop new therapeutic strategies. All trypanosomatids depend on the same unusual mode of gene expression involving polycistronic transcription of protein coding genes and trans splicing of nuclear pre-mRNA. Of central importance to this process is the parasite-specific spliced leader (SL) RNA from which the 5'terminal part is cleaved and fused to the 5'end of each mRNA. Since SL RNA is consumed in this process, the parasites crucially depend on continuously strong SL RNA synthesis throughout their life cycle. We have recently identified and characterized several proteins in T. brucei which are essential for SL RNA gene transcription. These include extremely divergent homologues of the general transcription factors TFIIA, TFIIB, TFIIH, and a candidate TFIIE suggesting that the parasites form a class II transcription pre-initiation complex (PIC) at the SL RNA gene (SLRNA) promoter to recruit RNA polymerase II for accurate transcription initiation. The extraordinary level of sequence divergence between these proteins and their mammalian orthologues, which prevented their identification in the completed Tritryp genome projects, indicates that the trypanosome PIC deviates substantially from its human counterpart. To explore this possibility, we will functionally characterize the PIC which forms at the SLRNA promoter. This will include RNA interference, in vitro transcription experiments and DNA-protein interaction assays to evaluate the identified proteins'importance for parasite growth and role in SLRNA transcription. Finally, we will analyze if the parasites use PIC formation to recruit RNA polymerase II to their long tandem arrays of protein coding genes. Chromatin immunoprecipitation in combination with deep sequencing will identify non-SLRNA PIC formation sites in the T. brucei genome. Together, these experiments may uncover unique and essential structural and/or biochemical features in a fundamentally important process, namely the recruitment of RNA polymerase II to DNA.
The proposed studies of this application will explore the essential process of accurate transcription initiation in trypanosomatid parasites which cause major human diseases. The proteins involved in this process exhibit an unprecedented divergence level to their human counterparts which raises the possibility that we can identify new targets for chemotherapeutic intervention of the parasites. Such targets are urgently needed because drugs for these diseases are few and toxic and parasite resistance to these drugs is on the rise.
