Disease-associated mutations in mitochondrial precursor tRNAs affect binding, m1R9 methylation, and tRNA processing by mtRNase P

Disease-related tRNA mutations affect binding affinity and 5′ end pre-tRNA processing activity by the mtRNase P. (A) Binding affinities of mutant mitochondrial pre-tRNAs for mtRNase P using standard binding conditions. 20 nM fluorescently labeled substrates were preincubated with 150 nM MRPP1/2 for 5 min and then titrated with 0–5 µM MRPP3 as described in Karasik et al. (2019). Data were collected when binding reached equilibria from changes in fluorescence anisotropy. The K_D values are calculated from the fit of the binding isotherm to the data and reported values are divided by the wild-type K_D values. (B) Single-turnover cleavage rates of mitochondrial pre-tRNAs containing mutations catalyzed by mtRNase P using standard cleavage conditions. Reactions contained 20 nM fluorescently labeled substrate, 1 µM MRPP3, and 0.4 µM MRPP1/2. The k_obs,cleavage values are calculated from the fit of the single exponential to the data and reported values are divided by the wild-type k_obs,cleavage values.