ABSTRACTParasitic protist Trypanosoma brucei causes African human and animal trypanosomiasis, a spectrum of diseasesaffecting the population and economy in sub-Saharan Africa. These digenetic hemoflagellates belong toKinetoplastea, a taxonomic class distinguished by possession of a kinetoplast. This nucleoprotein body containsmitochondrial DNA (kDNA) of two kinds: ~25 maxicircles (each ~23 kb) encoding rRNAs, ribosomal proteins andsubunits of respiratory complexes, and approximately 5000 of ~1 kb minicircles bearing guide RNA genes.Relaxed maxicircles and minicircles are interlinked and packed into a dense disc-shaped network by associationwith histone-like proteins. Decades of kDNA studies have unraveled fascinating phenomena of general biologicalsignificance, such as DNA bending and mRNA editing, and revealed exquisite details of replication and RNAprocessing. However, the molecular mechanisms of transcription remain virtually unexplored and arguablyconstitute the most critical gap in understanding mitochondrial gene expression. The historically enduring viewof polycistronic RNA synthesis has abridged efforts to investigate transcription’s contribution to regulatinggenome activity. In contrast, this proposal presents evidence that maxicircle and minicircle genes are individuallytranscribed into 3? extended precursors. The transcription start site defines pre-mRNA 5? terminus, which issubsequently converted into monophosphorylated state by a pyrophosphohydrolase complex, termed thePPsome. Most guide RNAs lack PPsome recognition sites and remain triphosphorylated. Furthermore, weestablish that antisense transcripts delimit the 3? boundaries of mature RNAs by blocking 3?-5? degradation ofprecursors by the 3? processome (MPsome). It follows that transcription start sites on sense and antisensestrands define 5? and 3? mRNA termini, respectively. These findings support a concept of mitochondrial gene-specific transcriptional control with broad implications in parasite development and pathogenesis. We posit thatelucidating transcription complex composition, DNA template requirements and functions of specific factors willbuild a foundation for this nascent research area. We propose to: 1) Characterize RNA polymerase complexfrom bloodstream and insect parasite forms, and assess transcription factors’ contributions to RNA synthesis; 2)Map maxicircle and minicircle promoters; and 3) Reconstitute the active transcription complex.