ABSTRACT Parasitic protist Trypanosoma brucei causes African human and animal trypanosomiasis, a spectrum of diseases affecting the population and economy in sub-Saharan Africa. These digenetic hemoflagellates belong to Kinetoplastea, a taxonomic class distinguished by possession of a kinetoplast. This nucleoprotein body contains mitochondrial DNA (kDNA) of two kinds: ~25 maxicircles (each ~23 kb) encoding rRNAs, ribosomal proteins and subunits 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 association with histone-like proteins. Decades of kDNA studies have unraveled fascinating phenomena of general biological significance, such as DNA bending and mRNA editing, and revealed exquisite details of replication and RNA processing. However, the molecular mechanisms of transcription remain virtually unexplored and arguably constitute the most critical gap in understanding mitochondrial gene expression. The historically enduring view of polycistronic RNA synthesis has abridged efforts to investigate transcription's contribution to regulating genome activity. In contrast, this proposal presents evidence that maxicircle and minicircle genes are individually transcribed into 3′ extended precursors. The transcription start site defines pre-mRNA 5′ terminus, which is subsequently converted into monophosphorylated state by a pyrophosphohydrolase complex, termed the PPsome. Most guide RNAs lack PPsome recognition sites and remain triphosphorylated. Furthermore, we establish that antisense transcripts delimit the 3′ boundaries of mature RNAs by blocking 3′-5′ degradation of precursors by the 3′ processome (MPsome). It follows that transcription start sites on sense and antisense strands 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 that elucidating transcription complex composition, DNA template requirements and functions of specific factors will build a foundation for this nascent research area. We propose to: 1) Characterize RNA polymerase complex from 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.