16] Isolation of mitochondrial tRNAs from human cells

Abstract

This chapter explains the isolation of mitochondrial tRNAs from human cells. Mammalian mitochondrial tRNAs possess many unique sequence, structural, and functional properties. All mammalian mitochondrial DNAs (mtDNAs) code for 22 tRNAs, which are sufficient for the translation of the mtDNA-encoded mRNAs, because a single tRNA is able to read all codons of a four-codon family. In contrast to prokaryotic, eukaryotic nuclear, and chloroplast tRNAs, whose structures are remarkably conserved, there are numerous sequence and structural alterations found among mammalian mitochondrial tRNAs. These include changes in highly conserved nucleotides or nucleotide base modifications, variations in the sizes of stem and loop structures, and alterations in conserved tertiary interactions. The chapter describes procedures for the isolation of single tRNA species based on a variety of fractionation methods. tRNA fraction, isolated by diethylaminoethyl (DEAE) cellulose, is subsequently further fractionated by electrophoresis through successive polyacrylamide gels. In addition, the chapter also describes procedure for cultured mammalian cells that allows the isolation of a highly purified total mitochondrial tRNA preparation. This procedure has been used previously to investigate the steady-state levels and metabolic properties of tRNAs isolated from HeLa cell mitochondria.