Abstract
Mutations in human mitochondrial isoleucine tRNA (hs mt tRNA(Ile)) are associated with cardiomyopathy and opthalmoplegia. A recent study showed that opthalmoplegia-related mutations gave rise to severe decreases in aminoacylation efficiencies and that the defective mutant tRNAs were effective inhibitors of aminoacylation of the wild-type substrate. The results suggested that the effectiveness of the mutations was due in large part to an inherently fragile mitochondrial tRNA structure. Here, we investigate mutant tRNAs associated with cardiomyopathy, and a series of rationally designed second-site substitutions introduced into both opthalmoplegia- and cardiomyopathy-related mutant tRNAs. A source of structural fragility was uncovered. An inherently unstable T-stem appears susceptible to misalignments. This susceptibility sensitizes both domains of the L-shaped tRNA structure to base substitutions that are deleterious. Thus, the fragile T-stem makes the structure of this human mitochondrial tRNA particularly vulnerable to local and distant mutations.
Highlights
The canonical tRNA cloverleaf folds into a two-domain Lshaped structure
Because the binding affinities of the mutated tRNAs for the enzyme were not affected, the mutants were effective inhibitors of the charging of wild-type tRNA. The aminoacylation of these mutant tRNAs was restored with compensatory mutations that reintroduced base pairing. These results prompted the proposal that the structure of the hs mt tRNAIle was inherently fragile and, small perturbations introduced by the pathogenic mutations were magnified into large losses in function and the creation of effective inhibitors
Aminoacylation Efficiency of a Cardiomyopathy-related Mutant tRNA—Mutants of hs mt tRNAIle associated with cardiomyopathy (Fig. 1) that involve substitutions in the T-stem and loop were generated by in vitro transcription and tested as substrates for the cognate recombinant hs mt isoleucyl-tRNA synthetase (IleRS). (Previous studies showed that transcripts of hs mt tRNAIle were relatively robust substrates for the recombinant enzyme, Ref. 11.) A tRNA containing an A59G mutation that alters an unpaired base in the T-loop had a significantly decreased rate of aminoacylation (Fig. 2)
Summary
The canonical tRNA cloverleaf folds into a two-domain Lshaped structure. One domain contains the amino acid attachment site and the 12-base pair acceptor-T⌿C minihelix. The minimized structures of hs mt tRNAs appear to be susceptible to point mutations, as errors in the corresponding mt genes are associated with disease. Because the binding affinities of the mutated tRNAs for the enzyme were not affected, the mutants were effective inhibitors of the charging of wild-type tRNA. The aminoacylation of these mutant tRNAs was restored with compensatory mutations that reintroduced base pairing. These results prompted the proposal that the structure of the hs mt tRNAIle was inherently fragile and, small perturbations introduced by the pathogenic mutations were magnified into large losses in function and the creation of effective inhibitors
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
