17] Analysis of aminoacylation of human mitochondrial tRNAs

Abstract

Publisher Summary This chapter describes several experimental approaches aimed at investigating the aminoacylation capacity of mitochondrial tRNAs. The analysis of human mitochondrial tRNAs has been hampered so far by the presence of excess of cytosolic tRNAs in the cell. Therefore, a specific strategy has to be designed for each tRNA, by choosing the experimental approach or combination of approaches most suitable in each case. Awareness is required for the multiple possible causes of misinterpretation; the chapter illustrates specific examples described that illustrate the advantages and limitations of each approach. As concerns in vitro aminoacylation, despite the use of a procedure that yields highly purified mitochondrial tRNA preparations, a minor and variable proportion of cytoplasmic tRNA contaminants may remain in these preparations. Cytoplasmic tRNA contaminating the mitochondrial fraction is expected not to be accessible to the mitochondrial synthetases and, therefore, not to be acylated, nor to compete with the acylation of mitochondrial tRNAs.