Trypanosome RNA Synthesis: Characterization of a Novel Transcription Factor as a

Villanova University, Villanova PA

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Abstract

Trypanosoma brucei is the causative agent of African Sleeping sickness. This parasitic infection compromises the lives of hundreds of thousands, and invariably results in death if untreated. There are few drugs available for treatment, and they can be toxic or even fatal. One area rich in potential therapeutic targets is trypanosome gene transcription. mRNA production in trypanosomes differs significantly from that of higher eukaryotes, which are the host for these infectious parasites. Particularly unusual in trypanosomes is RNA Polymerase II (RNAP II)-dependent gene transcription. Protein-coding genes are transcribed as long, polycistronic pre-mRNAs that are resolved into individual mature messages by the trans-splicing of a Spliced Leader (SL) RNA onto the 5? end and the addition of a poly (A) tail onto the 3? end of the primary mRNA transcript. As every functional protein-coding message contains an SL RNA, the synthesis of this RNA is essential for parasite survival. SL RNA synthesis is also RNAP II-dependent and our understanding of the associated proteins required for SL RNA synthesis and their mechanism of action has evolved over recent years, but is still far from complete. Our knowledge pertaining to the transcription of protein-coding genes in these parasites is scant. Transcriptional studies in trypanosomes have the potential to reveal novel biochemical pathways and processes. These processes, unique from those in man, could potentially provide new therapeutic targets. Studies of the trypanosome general transcription factor TFIIB revealed an interacting protein (TFIIB Associated Factor 49 KDa, TAF49) that is likely to function as a trypanosome-specific transcription factor in the synthesis of one or more RNAs. Our laboratory aims to characterize the role of this novel protein in transcription by using in vitro transcription reactions, chromatin immunoprecipitation (ChIP) studies, and by identifying and characterizing interacting protein-partners using techniques such as Tandem Affinity Purification (TAP). These experiments will contribute to our understanding of the mechanism of RNAP II-dependent gene transcription in these infectious eukaryotes, which will aid in our understanding of the survival tactics employed by these parasites and likely will uncover potential drug targets.