Identification and characterization of novel core promoter elements and a new transcription factor in Trichomonas vaginalis.

The molecular mechanisms involved in regulating eukaryotic gene expression have been primarily studied in fungi, plants and animals. By comparison, far less is known about the properties controlling gene expression of the more divergent protozoa. The studies presented in this dissertation focus on the protozoan parasite Trichomonas vaginalis and aim to identify novel eukaryotic gene expression mechanisms. Prior to these studies only one core promoter element, the initiator (Inr), had been identified in T. vaginalis protein coding genes, even though T. vaginalis encodes genes for almost the full compliment of general transcription factors shown to recognize the known metazoan core promoter elements. A novel initiator binding protein (IBP39) recognizes the T. vaginalis Inr. Structural studies have suggested that IBP39 can play a role in the recruitment of RNA polymerase II to the transcription pre-initiation complex through interactions between the C- terminal domains of the two proteins. We used genetic and biochemical analysis to further characterize the IBP39 C-domain and RNA polymerase II C-terminal domain (RNAP II CTD) interaction, and showed that the IBP39 C-domain and RNAP II CTD interact in vitro, mediated through hydrophobic interactions. Computational analysis of the ∼7X coverage of the T. vaginalis genome has predicted that ∼75 percent of putative core promoters are predicted to contain an Inr. The lack of an Inr in ∼25 percent of T. vaginalis core promoters begged the question of what other DNA elements are directing T. vaginalis transcription. Bioinformatics analysis of a protein-coding gene upstream region database did not reveal the obvious presence of functional known eukaryotic core promoter elements. However, two novel core promoter elements, Motif 3 (M3) and Motif 5 (M5), were identified. M3 and M5 are conserved in position relative to the transcription start site, and mutations within either motif significantly reduce transcriptional activity, showing that these two motifs are bona fide core promoter elements. A new transcription factor, M3 binding protein (M3BP), was then identified using M3-DNA affinity chromatography of T. vaginalis crude nuclear extract and MudPIT mass spectrometry. In addition to identifying novel eukaryotic gene expression properties, these studies provide the framework for additional T. vaginalis gene expression studies.