Trypanosomiasis and Leishmaniasis are major diseases in developing countries throughout the world. In these areas 100,000 people are currently infected with trypanosomes. Human illness includes African trypanosomiasis (""""""""sleeping sickness""""""""), Chagas' disease (endemic in regions of South and Central America) and cutaneous, mucocutaneous and visceral Leishmaniasis (endemic in parts of the Middle East and the Indian subcontinent). African trypanosomiasis is spread to humans by the bite of tsetse flies that harbor the Trypanosoma brucei subspecies. The disease burden is estimated by the World Health Organization to be two million Disability Adjusted Life Years. The cost of treatment is high, and untreated infection usually results in death. However, trypanosomatids share many metabolic pathways among themselves that differ enough from human pathways to be exploited in the development of therapeutics that are more efficacious than those currently available. One possible therapeutic approach is suggested by the observation that trypanosomatids have strikingly unusual mRNA biosynthetic pathways. The purpose of this proposal is to gain an understanding of how SL RNA and mRNAs are transcribed in trypanosomes. There appears to be extensive transcription in trypanosomes, but we have little information as to how mRNAs are produced. Therefore, the differences in mRNA maturation that exist among different mRNAs in one life cycle form may be regulated, in part, by differential mRNA synthesis. We have made major headway in the field of trypanosome gene expression by identifying and beginning to characterize Leptomonas seymouri PBP-1 and PBP-2. These proteins are among the first double stranded sequence-specific DNA binding proteins identified in trypanosomatids. They appear to function as basal transcription factors that regulate SL RNA expression; their role in pre-mRNA synthesis is unknown. We propose to understand to the structure and function of these proteins by characterizing in biochemical and genetic detail T. brucei PBP-1 and PBP-2. Having identified the trypanosomal SL RNA gene promoter as RNAP II-dependent, we hypothesize that PBP-1 and/or PBP-2 may function in other RNAP II-dependent synthesis, specifically pre-mRNA synthesis. We will directly test this possibility and identify additional transcription factors involved in RNAP II-dependent RNA synthesis by analyzing pre-RNA synthesis from a central region of T. brucei chromosome 2. We have recently developed a preinitiation complex formation assay, a Chromatin Immunoprecipation assay and a yeast two-hybrid system that are pertinent tools for our transcriptional dissections.
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