Molecular basis for mitochondrial transcription in Trypanosoma brucei

• Applicant: Professor Dr. Hauke Hillen
• Subject Area: Biochemistry ; Structural Biology ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 540361014

T. brucei is a single-celled protozoan parasite that belongs to the group of the Excavata, which causes severe diseases in humans and livestock. It harbours a single mitochondrion that contains a mitochondrial genome (kinetoplast DNA, kDNA) with unique topology, the expression of which is crucial to the parasitic life cycle. Recently, molecular insights into T. brucei mitochondrial translation have been obtained, which revealed a highly specialised mitoribosome and revealed the essential functional elements conserved throughout evolution. Like all eukaryotes, T. brucei also encodes for a specialised mitochondrial RNA polymerase (Tb-mtRNAP), which is thought to carry out transcription of the kDNA. To date however, no molecular or mechanistic information on the transcription apparatus in T. brucei mitochondria is available, and kinetoplast mitochondrial gene expression therefore remains poorly understood. Mitochondrial transcription has been studied most prominently in yeast and human cells, that only reflect the evolution of a single eukaryotic supergroup, the Opisthokonta. In order to understand how the modern-day mitochondrial gene expression system arose from the endosymbiotic event that occurred more than two billion years ago, we need to study organisms from distantly related eukaryotic supergroups. Our groups have previously extensively characterised the structure and function of the kDNA segregation machinery (Ochsenreiter lab) and the structural basis of transcription in human mitochondria (Hillen lab). Here, we propose a collaborative project that aims to elucidate the molecular basis of transcription in T. brucei mitochondria. To achieve this, we will combine our expertises in the in vivo cell biology of T. brucei (Ochsenreiter lab) and with the structural characterization of mitochondrial transcription complexes (Hillen lab) in order to gain a detailed understanding of this process from the cell biological to the molecular scale. In particular, we will (i) define the localization and interactome of Tb-mtRNAP, and identify potential additional mitochondrial transcription factors; (ii) investigate the consequences of loss of Tb-mtRNAP and associated factors in vivo; (iii) reconstitute T. brucei mitochondrial transcription in vitro from purified components and establish functional assays and (iv) determine structures of endogenous and reconstituted Tb-mtRNAP complexes by single-particle cryo-EM. Taken together, this collaborative project will provide the first comprehensive picture of transcription in kinetoplast mitochondria, which may provide a new avenue for drug development, as was just recently shown for the human mitochondrion.

• DFG Programme: Research Grants
• International Connection: Switzerland
• Cooperation Partner: Dr. Torsten Ochsenreiter