Mammalian Mitochondrial tRNAs: A Modified Nucleotide 3′ to the Anticodon May Modulate Their Codon Response

Abstract

Mammalian mitochondria contain a discrete, double-stranded DNA that encodes several proteins and the RNAs responsible for their synthesis. The complete nucleotide sequence of both the human and the bovine mtDNAs (Anderson et al. 1981) reveals the existence of 22 putative mitochondrial tRNA genes. Earlier, we reported the isolation and complete nucleotide sequence of one bovine mitochondrial tRNA (deBruijn et al. 1980), and now we report the results of studies showing that the 22 putative bovine mitochondrial tRNA genes are encoded into 22 mature, stable tRNA species and that the presence or absence of a modified nucleotide in the position immediately 3′ to the anticodon may modulate their codon response.

ISOLATION AND tRNA SEQUENCING
Bovine liver mitochondrial tRNAs were isolated and fractionated on two successive RPC-5 columns (Roe et al. 1973). Following further purification by polyacrylamide gel electrophoresis (deBruijn et al. 1980), their nucleotide composition was determined using the tritium borohydride method (Randerath et al. 1974). Complete nucleotide sequences were obtained by partial ribonuclease digestion of both the 3′ and the 5′ ³²P-postlabeled intact tRNAs (Chen and Roe 1980) and by the PEI-cellulose thin-layer chromatography (TLC) readout method (Gupta and Randerath 1979).

STRUCTURAL FEATURES OF THE BOVINE MITOCHONDRIAL tRNAS
Analysis of the complete nucleotide sequence of each bovine liver mitochondrial tRNA (see Fig. 1 for representative structures) reveals a much higher A + U content and a concomitantly lower G + C content than in their cytoplasmic counterparts (Gauss and Sprinzl 1981), as well as several other unorthodox features. The stem...