Mitochondrial transcription in Trypanosoma brucei: Conserved proteins and unusual initiations

Trypanosoma brucei has an extraordinary mitochondrial genome and is one of the earliest organisms to possess mitochondria. Mitochondrial gene expression in this parasitic protist is complex and developmentally regulated, but little is known about the transcription of this unusual kinetoplast DNA. In order to pinpoint promoter regions within the T. brucei mitochondrial genome, the 5' ends of the guide RNAs gMURF2-II and gCYb(560) were characterized. Each of these genes have uncommon kinetoplast DNA locations not typically associated with transcription initiation events. The gMURF2-II gene is located entirely within the 5' end of the ND4 gene. Interestingly, the gMURF2-II and ND4 transcripts are generated by distinctly different events; the ND4 mRNA is processed from a polycistronic precursor, while transcription of the gRNA initiates downstream of the 5' end of the ND4 gene. The data presented in this dissertation likewise show that the mature gCYb(560) gRNA is also a primary transcript and that the 5' end heterogeneity previously observed for this gRNA is a result of multiple transcription initiation sites and not imprecise 5' end processing. Together, these data indicate that gRNA genes represent individual transcription units regardless of genomic context and suggest a complex mechanism for mitochondrial gene expression in T. brucei.;In spite of its phylogenetic position near the acquisition of mitochondria by eukaryotes and its bizarre mitochondrial genome structure, the core components of mitochondrial transcription found in other eukaryotes are conserved in trypanosomes and their kin. The gene encoding the mitochondrial RNA polymerase, TbmtRNAP was cloned and shown to be developmentally regulated via differential RNA processing and stability. In addition, TbmtTFB, a homologue to the universally conserved mitochondrial transcription factor, mtTFB, was identified in trypanosomes although no homologue to the vertebrate factor TFAM was found. Over-expression of TbmtTFB caused a slight increase in growth rate, while RNAi against the gene resulted in slower growth. This together with the inability to generate double knockouts of this gene suggests an important role for this TbmtTFB in T. brucei , although the precise function of the protein is still unresolved.