A disease-causing point mutation in human mitochondrial tRNAMet results in tRNA misfolding leading to defects in translational initiation and elongation

Abstract

The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNAMet (hmtRNAMet) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNAMet with a C8. The hmtRNAMet serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U→C mutation leads to a failure of the tRNA to respond conformationally to Mg2+. This mutation results in a drastic disruption of the structure of the hmtRNAMet, which significantly reduces its aminoacylation. The small fraction of hmtRNAMet that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg2+, the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg2+, the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg2+-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.