Comparative analysis of the pathogenic mechanisms associated with the G8363A and A8296G mutations in the mitochondrial tRNA(Lys) gene

Biochem J. 2005 May 1;387(Pt 3):773-8. doi: 10.1042/BJ20040949.


Two mutations (G8363A and A8296G) in the mtDNA (mitochondrial DNA) tRNA(Lys) gene have been associated with severe mitochondrial diseases in a number of reports. Their functional significance, however, remains unknown. We have already shown that homoplasmic cybrids harbouring the A8296G mutation display normal oxidative phosphorylation, although the possibility of a subtle change in mitochondrial respiratory capacity remains an open issue. We have now investigated the pathogenic mechanism of another mutation in the tRNA(Lys) gene (G8363A) by repopulating an mtDNA-less human osteosarcoma cell line with mitochondria harbouring either this genetic variant alone or an unusual combination of the two mutations (A8296G+G8363A). Cybrids homoplasmic for the single G8363A or the A8296G+G8363A mutations have defective respiratory-chain enzyme activities and low oxygen consumption, indicating a severe impairment of the oxidative phosphorylation system. Generation of G8363A cybrids within a wild-type or the A8296G mtDNA genetic backgrounds resulted in an important alteration in the conformation of the tRNA(Lys), not affecting tRNA steady-state levels. Moreover, mutant cybrids have an important decrease in the proportion of amino-acylated tRNA(Lys) and, consequently, mitochondrial protein synthesis is greatly decreased. Our results demonstrate that the pathogenicity of the G8363A mutation is due to a change in the conformation of the tRNA that severely impairs aminoacylation in the absence of changes in tRNA stability. The only effect detected in the A8296G mutation is a moderate decrease in the aminoacylation capacity, which does not affect mitochondrial protein biosynthesis.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminoacylation
  • Cell Line, Tumor
  • DNA, Mitochondrial / genetics
  • Gene Expression Regulation / genetics*
  • Humans
  • MERRF Syndrome / genetics
  • MERRF Syndrome / physiopathology
  • Mitochondria / metabolism*
  • Mutation
  • Phenotype
  • Protein Conformation
  • RNA, Transfer, Lys / genetics*
  • RNA, Transfer, Lys / physiology


  • DNA, Mitochondrial
  • RNA, Transfer, Lys