The trypanosomatid parasites Trypanosoma brucei, T. cruzi and Leishmania spp. cause the major human diseases African Sleeping Sickness, Chagas' disease, and Leishmaniasis, respectively. Since drugs for these diseases are few, toxic and difficult to administer, and parasite resistance to these drugs is on the rise, it becomes increasingly important to develop new therapeutic strategies. While these parasites have developed unique host-parasite interactions, they all share the same unusual mode of gene expression involving polycistronic transcription of protein coding genes and trans splicing of nuclear pre-mRNA. A key molecule in this process is the 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 trans splicing, parasite viability crucially depends on continuously strong SL RNA synthesis. Therefore, inhibition of SL RNA gene (SLRNA) transcription appears to be a promising broadband strategy against trypanosomatid parasites. So far, we have been able to identify and characterize four transcription factors comprising 25 proteins in T. brucei that are essential for the process: the promoter-binding complex TRF4/SNAPc/TFIIA, TFIIB, a complex of TFIIH and TFIIE, and the trypanosome mediator. Our data show that these proteins form a transcription pre-initiation complex at the SLRNA promoter and recruit RNA polymerase II for accurate transcription initiation. Conversely, we found no evidence that TFIIB binds to divergent strand switch regions (dSSRs) known to initiate RNA polymerase II transcription of the protein coding gene arrays. We therefore propose in Aim 1 to continue our biochemical characterization of RNA polymerase II transcription factors to identify the most promising targets for further analysis; besides the specified factors, these include new proteins that co-purified with RNA pol II as well as a novel TFIIA- associated complex and a CDK-related kinase both of which appear to function specifically in pre-mRNA synthesis. For these factor characterizations we plan to employ a plethora of genetic and biochemical experiments which include tandem affinity purification of protein complexes, conditional gene silencing experiments and in vitro transcription assays.
In Aim 2, we will use a systematic approach to investigate the mechanism of transcription initiation in dSSRs which will be based on a genome-wide analysis of RNA polymerase II occupancy and on a mutational analysis of dSSR-driven reporter gene expression. Overall, these experiments may uncover unique and essential factors or factor domains in a fundamentally important process, namely the recruitment of RNA polymerase II to DNA. Moreover, they will provide a mechanistic understanding of the first step in protein expression which will help to control trypanosomatids in the long term.
The proposed studies of this application will explore mechanism and relevant factors of RNA polymerase II transcription in Trypanosoma brucei, a member of a group of parasites that cause major, devastating diseases in humans. 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 this disease are few and toxic, and parasite resistance to existing drugs is on the rise. Moreover, a thorough understanding of trypanosome gene expression will help to control the parasite in the long term.
