Monday, November 25, 2024
11/25/2024
DESCRIPTION (provided by applicant): Trypanosome-related disease is responsible for significant devastation of productive human lives and high mortality rates throughout the world. The trypanosome transcriptional machinery appears to exhibit intriguing differences from the more conserved machineries of other, better- studied eukaryotes (yeast and mammals). For example, Trypanosoma brucei and other members of the trypanosomatid family contain an unusual RNA polymerase II enzyme and what appears to be an abridged set of transcription factors. Moreover, pre-mRNA start sites remain elusive and transcription initiation is not coordinated with mRNA 5' capping. These mechanistic differences provide a compelling argument for studies into the structure and function of RNA polymerase II- dependent processes in trypanosomes, with the ultimate goals of enhancing our understanding of this evolutionarily diverse process, and developing organism-specific chemotherapies. Although our ultimate objective is to understand the mechanism of RNA polymerase II- dependent gene expression, our short-term goals will focus on characterizing the proteins and sequences that are known, or suspected, to function in pre-mRNA synthesis. To date, we have shown that tSNAPc, tTBP, TFIIB, TFIIA-gamma and RNA polymerase II assemble at the SL RNA gene promoter and are necessary to direct SL RNA synthesis. Because RNA polymerase II is also required for pre-mRNA synthesis, the current proposal will extend analyses of these proteins, as well as several novel RNA polymerase II-interacting proteins recently identified in our lab, to genomic loci thought to direct pre-mRNA expression. The mechanism of pre-mRNA gene expression will be investigated using the following two complementary approaches. First, the function of purified protein factors and DNA sequences will be determined by in vitro transcription studies developed in several laboratories, including our own, in combination with protein-protein and protein-DNA interaction studies. A second approach will exploit in vivo studies, originally developed by others, to assess the function of each protein in cell viability and pre-mRNA expression. These studies are expected to provide us with interesting new insights into the diverse mechanisms by which eukaryotes accomplish fundamentally similar ends, as well as potential targets for drug therapy against what remains a wide-spread and crippling pathogen. PUBLIC HEALTH RELEVANCE: Pathogenic trypanosomes are a major, worldwide public health scourge. In Africa, Trypanosoma brucei ssp. infect humans and domestic cattle, causing African sleeping sickness and Nagana, respectively. Human infection by the subspecies T. b. gambiense and rhodesiense are responsible for almost 2 million disability-adjusted life-years and an estimated 48,000 deaths each year. In the Western hemisphere, trypanosome infection causes fatal enlargement of the colon, as well as serious cardiomyopathy known as Chagas' disease. Approximately 11 million people are afflicted with Chagas' disease and 45,000 people die yearly. In South Asia, South America, and the Middle-East, a closely related group of parasites, the Leishmania, cause debilitating and often fatal Leishmaniasis. Not surprisingly, this disease is a problem for American troops in Iraq and Afghanistan; more than 700 cases have been diagnosed in returning military personnel. At present, there are no vaccines to protect humans from trypanosomal infections and current drugs are toxic, ineffective and expensive. The goal of our biomedical work is to define unique features of trypanosome biology and then to exploit these traits for the design of anti-parasitic chemotherapies.
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