In vitro mutagenesis of the mitochondrial leucyl-tRNA synthetase of S. cerevisiae reveals residues critical for its in vivo activities

Abstract

The mitochondrial leucyl-tRNA synthetase (mLRS) of Saccharomyces cerevisiae is involved in both mitochondrial protein synthesis and pre-mRNA splicing. We have created mutations in the regions HIGH, GWD and KMSKS, which are involved in ATP-, amino acid- and tRNA-binding respectively, and which have been conserved in the evolution of group I tRNA synthetases. The mutants GRD and NMSKS have no discernible phenotype. The mutants AWD and ARD act as null alleles and lead to the production of 100% cytoplasmic petites. The mutants HIGN, NIGH and KMSNS are unable to grow on glycerol even in the presence of an intronless mitochondrial genome and accumulate petites to a greater extent than the wild-type but less than 40%. Experiments with an imported bI4 maturase indicate that the lesion in these mutations primarily affects the synthetase and not the splicing functions.

The phenotypic expression of mitochondrial tRNA-mutations can be modulated by either mitochondrial leucyl-tRNA synthetase or the C-terminal domain thereof

“…Class I aaRSs are specific for amino acids Val, Leu, Ile, Met, Cys, Glu, Gln, Tyr, Trp, and Arg. Their active site is located in a Rossman fold nucleotide-binding catalytic domain (made of six parallel β-strands alternating to α-helices; Li et al, 1992 ). Class II aaRSs are specific for amino acids Gly, Ala, Ser, Thr, Asn, Asp, Lys, His, Phe, and Pro.…”

The isolated carboxy‐terminal domain of human mitochondrial leucyl‐tRNA synthetase rescues the pathological phenotype of mitochondrial tRNA mutations in human cells

“…Furthermore, aaRSs are grouped in two classes, based on their structural and functional properties (Ibba & Soll, ; Antonellis & Green, ; Suzuki et al , ). Class I aaRSs (specific for aminoacids Val, Leu, Ile, Met, Cys, Glu, Gln, Tyr, Trp and Arg) are mostly monomeric and contain a nucleotide‐binding catalytic domain with a Rossman fold, made of six parallel β‐strands alternating to α‐helices (Li et al , ; Martinis & Boniecki, ). They approach the end of the tRNA acceptor helix from the minor groove side and catalyse the attachment of the aminoacid to the 2′‐OH at the 3′‐end of the tRNA chain.…”

Genetic Evidence for Interaction between Cbp1 and Specific Nucleotides in the 5′ Untranslated Region of Mitochondrial Cytochrome b mRNA in Saccharomyces cerevisiae

“…Expression of COB requires participation of nuclearly encoded proteins. For example, Cbp2 (28,42), Mrs1 (5,21,22), Mrs2 (20,43), and Nam2 (24,26) are required for intron splicing; Cbp1 stabilizes COB mRNA (8,12); and Cbs1 (29,36) and Cbs2 (Cbp7) (29,34,35) are COB-specific translation factors. None of these nuclearly encoded factors is essential for growth of yeast on fermentable carbon sources (e.g., glucose), but strains with null mutations in the genes encoding these factors have a PET (petite) phenotype; they form smaller colonies than the wild-type strains on glucose media.…”