Nucleotide sequence and functional characterization of a mitochondrial tRNATrpfromTetrahymena thermophila

The use of the termination codon UGA as a tryptophan codon in mitochondria appears to be one of the most common deviations from the standard genetic code. This change in the mitochondrial genome is accomodated by the presence of an anticodon U*CA in mitochondrial (mt) tRNAP (1). Recently a tRNA gene with a TCA anticodon has been identified in the mt genome of the ciliates Poramecium tetraurelia (2) and Tetrahymena pyriformis (3). Here we describe a mt tRNAP with the anticodon U*CA from Tetrahymena thermophila. Total tRNA and aminoacyl-tRNA synthetase were isolated from the cells of T. thermophila as described previously (4). Fractionation of tRNAs by BD-cellulose column chromatography, purification of tRNAs by polyacrylamide gel electrophoresis and sequencing of tRNA by post-labelling techniques was carried out according to Beier et al. (5). UGA suppressor activity of T. thermophila column or gel fractions lwas assayed in a reticulccyte lysate programmed with globin mRNA (6). Only one tRNA species capable of suppressing the UGA termination codon in /3-globin mRNA was identified in fractions of total Tetrahymena tRNA. This tRNA is charged with tryptophan by a crude Tetrahymena aminoacyl-tRNA synthetase preparation. The nucleotide sequence of the tRNAP is shown in Fig. 1. It exhibits a high degree of homology with the known sequence of the mt tRNA? gene from T. pyriformis (3), differing only in two nucleotides at positions 16 and 69. Consequently, we conclude that we have identified the corresponding T. thermophila tRNA with a U*CA anticodon. The modified undine (U*) in position 34 is very likely 5[[(carboxymethyl)amino]methyl]uridine (cmnmU) as indicated by its thin-layer chromatographic mobility in two solvent systems (4). U55 is only partially modified to pseudouridine. A* is probably N-isopentenyladenosine (iA) or 2-methylthio-N-isopentenyladenosine (msiA). The Tetrahymena mt tRNAP (U*CA) functions in cytoplasmic protein synthesis as demonstrated in vitro by the efficient suppression of the UGA termination codon of 0-globin mRNA in a reticulocyte lysate (Fig. 2). However, the suppression is strictly dependent on the simultaneous presence of aminoacyltRNA synthetase from Tetrahymena cells. REFERENCES