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POLRMT is the dedicated mitochondrial RNA polymerase in metazoans and is essential for priming mitochondrial DNA (mtDNA) synthesis. Aberrant POLRMT causes mitochondrial dysfunction and has previously been linked to human metabolic disease. The small molecule IMT1B is an allosteric inhibitor of POLRMT, and its use in diverse model organisms is informative about various aspects of mtDNA synthesis and transcription. Previously, this drug has been shown to be effective in perturbing mtDNA gene expression in human cells and mice. Moreover, a leucine to phenylalanine substitution at position 813 of human POLRMT is predicted to disrupt interaction with the drug. However, the effect of the F813L mutation on the efficacy of POLRMT inhibition (POLRMTi) has not been rigorously tested. Here, we determined by multiple sequence alignment and phylogenetic analysis that this mutation in POLRMT is common among invertebrates, including the model nematode Caenorhabditis elegans. AlphaFold analyses of metazoan POLRMT folding suggest that the F813L substitution alters the physicochemical features of the IMT1B binding pocket. Finally, we find that IMT1B treatment of larval Caenorhabditis elegans has little impact on mtDNA copy number, suggesting that POLRMTi via IMT1B may not be effective in this model organism. 

This protocol details a method for 5-ethynyl uridine labeling of nascent mitochondrial genome transcription coupled with immunofluorescence detection of mtDNA-binding proteins in cultured cells. The result is a fluorescent readout of localized mitochondrial gene transcription compatible with high-resolution microscopy. 

Eve Kakudji and Samantha Lewis discuss the structure and function of mitochondrial nucleoids - large nucleoprotein complexes containing mitochondrial DNA and the regulatory factors necessary for its packaging, replication, transcription, and repair. 

This protocol describes the metabolic labeling of mitochondrial translation in cultured cells (such as IMR90) using an amino acid L-Homopropargylglycine (L-HPG).