Human mitochondrial TyrRS disobeys the tyrosine identity rules

Abstract

Human tyrosyl-tRNA synthetase from mitochondria (mt-TyrRS) presents dual sequence features characteristic of eubacterial and archaeal TyrRSs, especially in the region containing amino acids recognizing the N1-N72 tyrosine identity pair. This would imply that human mt-TyrRS has lost the capacity to discriminate between the G1-C72 pair typical of eubacterial and mitochondrial tRNATyr and the reverse pair C1-G72 present in archaeal and eukaryal tRNATyr. This expectation was verified by a functional analysis of wild-type or mutated tRNATyr molecules, showing that mt-TyrRS aminoacylates with similar catalytic efficiency its cognate tRNATyr with G1-C72 and its mutated version with C1-G72. This provides the first example of a TyrRS lacking specificity toward N1-N72 and thus of a TyrRS disobeying the identity rules. Sequence comparisons of mt-TyrRSs across phylogeny suggest that the functional behavior of the human mt-TyrRS is conserved among all vertebrate mt-TyrRSs.

FIGURE 1.

Sequence comparison of the clusters in the catalytic domains of TyrRSs in the vicinity to identity elements in tRNA^Tyr. The figure emphasizes the clusters of human mt-TyrRS and compares their sequences to those of TyrRSs from four phylogenetic groups ranked according to the nature of the N1-N72 base pair. For each group (eubacteria, mitochondria—but without vertebrate mitochondria—archaea, eukarya) three sequences are explicitly given. Among mitochondrial TyrRSs, those of Arabidopsis thaliana and Caenorhabditis elegans were annotated for the purpose of this work. Alignments were done with Tcoffee (Poirot et al. 2003) or 3DCoffee (Poirot et al. 2004) to take into account the known TyrRS crystallographic structures (T. thermophilus, Yaremchuk et al. 2002; M. jannaschii, Kobayashi et al. 2003; Bacillus stearothermophilus, Brick et al. 1989; Staphylococcus aureus, Qiu et al. 2001; and Homo sapiens mini-TyrRS, Yang et al. 2002). Numberings flanking the alignments correspond to those of TyrRS from T. thermophilus for eubacterial/mitochondrial TyrRSs and from M. jannaschii for archaeal/eukaryal TyrRSs; numbering of human mt-TyrRS is also given. The asterisks below the human mitochondrial sequence indicate strict conservation of amino acids in vertebrate mt-TyrRSs (see the text). Consensus sequences were established with up to 31 known or predicted sequences (exact numbers are indicated in parentheses); the displayed consensus residues are present in >70% of the analyzed sequences. Underlined amino acids are strictly conserved; amino acids with the same properties are depicted by (φ) for hydrophobic, (+) positively charged, (−) negatively charged, (a) aliphatic, (μ) small, and (h) with hydroxyl group. The consensus within the G1-C72 and C1-G72 groups and within the three domains of life or mitochondria are emphasized by colors (notice that the conserved amino acids crossing the G1-C72/C1-G72 barrier are in green). Squared residues are those found in direct contact with bases of either T. thermophilus tRNA^Tyr (Yaremchuk et al. 2002) or M. jannaschi tRNA^Tyr (Kobayashi et al. 2003).

FIGURE 2.

Sequence of wild-type and mutated tRNA^Tyr molecules. The nature of the N1-N72 base pair is emphasized in each molecule. Wild-type human mt-tRNA^Tyr is depicted in A, its variant with the reversed C1-G72 pair in B, chimeric mt/cyt-tRNA^Tyr with cyt-tRNA^Tyr acceptor stem in C, and wild-type human cyt-tRNA^Tyr in D. The sequence of native tRNA^Tyr from E. coli (E) is displayed in italics. Mitochondrial and E. coli sequences are in gray whereas eukaryal (cytosolic) sequences are in black. Sequence data are taken from Sprinzl and Vassilenko (2005). All transcripts were produced in vitro using the “transzyme” method and purified as described in Fechter et al. (1998) whereas native tRNA^Tyr from E. coli (E) was from Subriden.