TRANSCRIPTIONAL CONTROL OF MITOCHONDRIAL GENE EXPRESSION IN TRYPANOSOMES

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.

Grant Number: 5R01AI152408-05
NIH Institute/Center: NIH
Principal Investigator: Ruslan Afasizhev