Extended Data Fig. 4: Identification of the IMT-binding pocket.
From: Small-molecule inhibitors of human mitochondrial DNA transcription
a, Schematic of the experimental workflow of the IMT-resistance screen using chemical mutagenesis. b, In vitro promoter-dependent transcription using the IMT-resistant L816Q and A821V POLRMT mutants in the presence of 0–10 μM IMT1B. A representative image from n = 2 independent experiments is shown. c, Quantification of b. Mean values of n = 2 independent experiments are shown. d, Inhibition of transcription initiation on IMT-resistant POLRMT mutants. In vitro transcription initiation was performed on linear LSP templates using wild-type and IMT-resistant POLRMT protein in the presence or absence of IMT1. A representative image from n = 2 independent experiments is shown. e, Increasing amounts of POLRMT do not rescue wild-type activity in the presence of IMT1B. In vitro promoter-dependent transcription on supercoiled circular LSP templates was performed in the presence of 10 μM IMT1B using increasing concentrations of wild-type or mutant POLRMT (L816Q or A821V). A representative image from n = 2 independent experiments is shown. f, Quantification of e. Mean values of n = 2 independent experiments are shown. g, Sanger sequencing of CRISPR–Cas9-engineered POLRMT mutations introduced into A2780 cells. h, Mitochondrial gene expression in POLRMT mutant cell lines (L796Q and L816Q, # denotes two independent clones). Data are expressed as fold of untreated wild-type control (DMSO) and given as mean ± s.e.m. (n = 7–9 biological replicates; two-way ANOVA, Sidak’s multiple comparisons test, DMSO–IMT1).
