This work seeks to understand how the unusual genetic code for methionine (Met) and isoleucine (Ile) in mammalian mitochondria is expressed. Mutations in mitochondrial tRNAMet and tRNAIle responsible for the expression are linked to Leber's hereditary optic neuropathy and numerous mitochondrial myopathies and neuromuscular disorders. A unique feature of the mitochondrial code is the assignment of the AUA codon to Met instead of Ile. This addition of a second codon for Met, in addition to the standard AUG codon, appears to be an adaptive mechanism to increase Met contents in proteins in response to the highly oxidative environment of mammalian mitochondria. It is accomplished by the presence of a novel 5-formyl modification to C34 (f5C34) in the wobble position of the CAU anticodon of mitochondrial tRNAMet and by the lack of modification in the GAU anticodon of mitochondrial tRNAIle, which reads only AUU and AUC for Ile. A multidisciplinary approach, consisting of enzymology, bioinformatics, protein purification, and kinetic analysis, will be used to elucidate the molecular mechanisms that ensure accurate expression of Met and Ile in mammalian mitochondria.
In aim 1, a combination of chemical synthesis and biochemical analysis of potential one-carbon donors of the 5-formyl group is undertaken to determine the biosynthetic pathway to f5C34. Enzymes involved in the pathway will be identified using a combination of protein purification, comparative genomics, and biochemical analysis of candidate gene products. This will open the way to study the mechanisms of these novel enzymes and to build bioinformatics models for determining their possible roles in mitochondrial tRNAMet-related diseases.
In aim 2, experiments will address how the status of anticodon modification in mitochondrial tRNAMet and tRNAIle determines the accuracy of the deviant code. Both the accuracy of tRNA aminoacylation, catalyzed by the mitochondrial Met- and Ile-tRNA synthetases, and the accuracy of tRNA decoding, catalyzed by the mitochondrial ribosome, will be interrogated in a comprehensive enzymatic and kinetic approach. New assays will be developed to reconstitute the mitochondrial ribosome and to investigate the decoding step, thus establishing an in vitro translation system similar to the one used in studies of the bacterial ribosome. These experiments are designed with a view towards a quantitative understanding of the accuracy of mitochondrial gene expression. The significance of the work is high in that it addresses at the fundamental level why mutations and lack of modification in mitochondrial tRNAs are associated with over 100 mitochondrial disorders.
The importance of the mammalian mitochondrial genetic code to public health is based on the discovery of over 100 human genetic diseases associated with mitochondrial disorders. The majority of these diseases are linked to mutations in mitochondrial tRNAs or to deficiencies in their nucleotide modifications. This work seeks to understand the molecular mechanisms responsible for the unusual decoding of human mitochondrial tRNAMet and tRNAIle in the expression of the mitochondrial code, with direct relevance to mitochondrial myopathies, encephalomyopathy, hypertension and hypertrophic cardiomopathy, and Leber's hereditary optic neuropathy.
| Peacock, Jacob R; Walvoord, Ryan R; Chang, Angela Y et al. (2014) Amino acid-dependent stability of the acyl linkage in aminoacyl-tRNA. RNA 20:758-64 |
| Hou, Ya-Ming; Yang, Xiaolu (2013) Regulation of cell death by transfer RNA. Antioxid Redox Signal 19:583-94 |
