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ESR 7 Project: Mechanisms and regulation of transcription termination in human mitochondria.

Partner: Maria Falkenberg

Institution: University of Gothenburg, Sweden

Duration: 36 months

Objectives: We have previously demonstrated that mitochondrial transcription is specifically terminated at a conserved sequence element denoted CSB II (Pham et al, J Biol Chem 2006;281:24647-52). During transcription of the G-rich CSB II sequence (5´-GGG GGG AGG GGG-3ʹ), a G-quadruplex structure is formed in the nascent RNA strand. The G-quadruplex structure stimulates premature termination of transcription, probably by weakening RNA-to-polymerase contacts, which in turn destabilizes the elongation complex. G-quadruplexes are four-stranded structures that can form in guanine-rich sequences by the stacking of planar G tetrads or quartets. The structures are very stable and occur in the human genome at e.g. telomeres. The transcription termination mechanism that we have identified is highly reminiscent of Rho-independent transcription termination in bacteria, with the exception that a G-quadruplex structure replaces the hairpin structure normally observed in thus type of bacterial transcription termination (Wanrooij et al, Proc Natl Acad Sci U S A. 2010;107:16072-7). In the planned project, we will investigate if the mechanism for transcription termination identified at CSB II also operates at other sites in the mitochondrial genome. We will map transcription termination sites and we will in collaboration with James Stewart, Max-Planck Institute for Biology of Ageing in Cologne, examine the sequence and secondary structural requirements of identified termination sites. Initial bioinformatics analyses of mtDNA, show that sequences similar to the known CSB II transcription termination site can be identified at locations at which transcription is expected to terminate. Please note that mammalian mitochondria cannot be transfected, so effects of mtDNA mutations must be investigated in vitro. Transcription termination at CSB II and initiation of mtDNA synthesis is a regulated process. Whereas about 60% of all transcription events terminates at CSB II in vitro, the fraction of termination events in a mitochondrial extracts are far lower, down to 1-2%. We have demonstrated that this effect is attributed to TEFM, a transcription elongation factor (Posse et al Nucleic Acids Res. 2015;43:2615-24.). The activity and the molecular mechanisms of this factor will be characterized in our reconstituted in vitro transcription system. We will also establish how TEFM affects replication initiation in mitochondria, reconstitute mtDNA replication initiation reaction in vitro and use mitochondrial extracts to search for additional factors that may influence this process. The in vivo role for TEFM and additional factors identified in the regulation of mitochondrial transcription will be addressed by establishing a cellular knock-out models using the CRISPR/Cas9 system.

Expected results: This project will further investigate the transcription termination mechanism in the mitochondrial genome. Furthermore, we will gain critical insights into the function of TEFM. The role of this factor will enhance our understanding of the regulation of mitochondrial transcription.