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 tRNA Met (hmtRNA Met ) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNA Met with a C8. The hmtRNA Met 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 8U3 C mutation leads to a failure of the tRNA to respond conformationally to Mg 2؉ . This mutation results in a drastic disruption of the structure of the hmtRNA Met , which significantly reduces its aminoacylation. The small fraction of hmtRNA Met 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 Mg 2؉ , the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg 2؉ , the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg 2؉ -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.Human mitochondria are subcellular organelles that produce more than 90% of the energy required by the cell. The mitochondrial genome encodes 13 proteins necessary for energy production, two rRNAs and all of the 22 tRNAs required for the synthesis of these proteins (1, 2). Mammalian mitochondrial tRNAs have several unusual features that distinguish them from canonical tRNAs. In many cases, they lack a number of the conserved or semi-conserved nucleotides that play important roles in creating the L-shaped tertiary structure of prokaryotic and eukaryotic cytoplasmic tRNAs (3). There is little detailed structural information on these tRNAs. No data are currently available that examine the structure of mammalian mitochondrial tRNAs with single nucleotide resolution. However, chemical and enzymatic probing has lead to the idea that these tRNAs have retained the basic cloverleaf structure of canonical tRNAs but that they lack several conserved tertiary interactions leading to a weaker three-dimensional structure (4 -8). In particular, a number of the long range interactions between the Dand T-arms of the tRNAs appear to be missing.All 22 tRNAs that function in mammalian mitochondria are encoded in the mitochondrial DNA. Considerable interest in mitochondrial tRNAs centers on the occurrence of diseases arising from mutations in their genes that lead to maternally inherited genetic diso...

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