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Updated: Apr 23, 2026
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
Biochemical characterization of pathogenic mutations in human mitochondrial methionyl-tRNA formyltransferase
Akesh Sinha1, Caroline Köhrer1, Michael H W Weber1
1From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Abstract:
N-Formylation of initiator methionyl-tRNA (Met-tRNA(Met)) by methionyl-tRNA formyltransferase (MTF) is important for translation initiation in bacteria, mitochondria, and chloroplasts. Unlike all other translation systems, the metazoan mitochondrial system is unique in using a single methionine tRNA (tRNA(Met)) for both initiation and elongation. A portion of Met-tRNA(Met) is formylated for initiation, whereas the remainder is used for elongation. Recently, we showed that compound heterozygous mutations within the nuclear gene encoding human mitochondrial MTF (mt-MTF) significantly reduced mitochondrial translation efficiency, leading to combined oxidative phosphorylation deficiency and Leigh syndrome in two unrelated patients. Patient P1 has a stop codon mutation in one of the MTF genes and an S209L mutation in the other MTF gene. P2 has a S125L mutation in one of the MTF genes and the same S209L mutation as P1 in the other MTF gene. Here, we have investigated the effect of mutations at Ser-125 and Ser-209 on activities of human mt-MTF and of the corresponding mutations, Ala-89 or Ala-172, respectively, on activities of Escherichia coli MTF. The S125L mutant has 653-fold lower activity, whereas the S209L mutant has 36-fold lower activity. Thus, both patients depend upon residual activity of the S209L mutant to support low levels of mitochondrial protein synthesis. We discuss the implications of these and other results for whether the effect of the S209L mutation on mitochondrial translational efficiency is due to reduced activity of the mutant mt-MTF and/or reduced levels of the mutant mt-MTF.
Insights
Mitochondrial translation requires N-formylation. Mutations in human mitochondrial methionyl-tRNA formyltransferase (mt-MTF) cause Leigh syndrome by reducing protein synthesis efficiency. Residual activity in S209L mutants supports low-level function.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- N-formylation of initiator methionyl-tRNA (Met-tRNA(Met)) by methionyl-tRNA formyltransferase (MTF) is crucial for translation initiation in bacteria, mitochondria, and chloroplasts.
- Metazoan mitochondrial translation uniquely employs a single tRNA(Met) for both initiation and elongation, with a portion being formylated for initiation.
- Compound heterozygous mutations in the nuclear gene for human mitochondrial MTF (mt-MTF) have been linked to reduced mitochondrial translation efficiency, oxidative phosphorylation deficiency, and Leigh syndrome.
Purpose of the Study:
- To investigate the functional impact of specific mutations (S125L and S209L) in human mt-MTF on its enzymatic activity.
- To compare the effects of human mt-MTF mutations with corresponding mutations (A89L and A172L) in Escherichia coli MTF.
- To understand the contribution of residual mt-MTF activity to mitochondrial protein synthesis in patients with these mutations.
Main Methods:
- Enzymatic assays were performed to quantify the activity of wild-type and mutant human mt-MTF (S125L, S209L).
- Corresponding mutations were introduced into E. coli MTF (A89L, A172L) to assess conserved functional effects.
- Analysis of residual enzyme activity in relation to observed clinical phenotypes in patients.
Main Results:
- The human mt-MTF S125L mutant exhibited a 653-fold reduction in activity compared to wild-type.
- The human mt-MTF S209L mutant showed a 36-fold reduction in activity.
- Both patients with compound heterozygous mutations rely on the residual activity of the S209L mutant to sustain minimal mitochondrial protein synthesis.
Conclusions:
- Mutations at Ser-125 and Ser-209 significantly impair human mt-MTF activity, explaining the observed mitochondrial dysfunction and Leigh syndrome.
- The S209L mutation retains partial activity, which is essential for supporting low levels of mitochondrial protein synthesis in affected individuals.
- The clinical impact of the S209L mutation likely results from a combination of reduced enzyme activity and potentially altered protein levels.
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