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 8U-->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.
Highlights
Human mitochondria are subcellular organelles that produce more than 90% of the energy required by the cell
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)
The diseases associated with mitochondrial tRNA mutations may arise from failure in the processing of the tRNA [13], from reduced stability of the tRNA [14, 15], from a reduction in aminoacylation [12, 16, 17], from a reduced ability of the mutated aminoacyl-tRNA to interact with mitochondrial elongation factor Tu (EF-Tumt)3 [16], and from the failure of the tRNA to be correctly modified leading to translational defects [18]
Summary
RNA Synthesis—Human mitochondrial tRNAMet transcripts for aminoacylation experiments were prepared by in vitro transcription using the hammerhead ribozyme construct described previously [23]. Reaction mixtures (100 l) contained 50 mM Tris-HCl, pH 7.6, 2.5 mM MgCl2, 2.5 mM ATP, 0.2 mM spermine, 200 g/ml bovine serum albumin, 0.2 units/l SUPERase1⁄7In RNase inhibitor, 40 M [35S]methionine (4,000 cpm/pmol), 50 nM human mitochondrial MetRS or 8 nM E. coli MetRS, and 1 M U8 or 8U3 C hmtRNAMet. The amount of aminoacylated tRNA formed was determined by trichloroacetic acid-precipitable counts at the indicated times. Formylation of Human Mitochondrial Met-tRNAMet— Formylation reactions (5 l) contained 20 mM Tris-HCl, pH 7.6, 100 M EDTA, 150 mM KCl, 7 mM MgCl2, 10 mM ME, 125 M folinic acid (Sigma), 100 nM normal or 8U3 C mutated [35S]Met-hmtRNAMet and 8 nM MTFmt. Reactions were performed at 37 °C for 0 – 8 min (0-min time point was taken in the absence of enzyme). 6 mM Mg2ϩ was added to the U8 and 8U3 C transcripts, and the UV-monitored thermal denaturation experiments were repeated
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