Dual targeting of isoleucyl-tRNA synthetase in Trypanosoma brucei is mediated through alternative trans-splicing.

Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNAs with their cognate amino acids. They are an essential part of each translation system and in eukaryotes are therefore found in both the cytosol and mitochondria. Thus, eukaryotes either have two distinct genes encoding the cytosolic and mitochondrial isoforms of each of these enzymes or a single gene encoding dually localized products. Trypanosomes require trans-splicing of a cap containing leader sequence onto the 5'-untranslated region of every mRNA. Recently we speculated that alternative trans-splicing could lead to the expression of proteins having amino-termini of different lengths that derive from the same gene. We now demonstrate that alternative trans-splicing, creating a long and a short spliced variant, is the mechanism for dual localization of trypanosomal isoleucyl-tRNA synthetase (IleRS). The protein product of the longer spliced variant possesses an amino-terminal presequence and is found exclusively in mitochondria. In contrast, the shorter spliced variant is translated to a cytosol-specific isoform lacking the presequence. Furthermore, we show that RNA stability is one mechanism determining the differential abundance of the two spliced isoforms.

Biochemical basis of mupirocin resistance in strains of Staphylococcus aureus.

Twenty one strains of Staphylococcus aureus, of varying resistance to mupirocin, were examined in order to determine the mechanism of resistance to this antibiotic; six of these strains were mupirocin sensitive (MIC 0.12-1.0 mg/L) nine moderately resistant strains (MIC 8-256 mg/L) and six highly resistant strains (MIC > 2048 mg/L). Mupirocin showed a time-dependent inhibition of the target enzyme, isoleucyl-tRNA synthetase (IRS); incubation of the antibiotic with this enzyme before adding the substrates markedly increased inhibition in sensitive strains. The IRS I50 values (the antibiotic concentrations which cause a 50% decrease in enzyme activity) correlated well with the MIC values for each strain (P < 0.01). The mean I50 value for sensitive strains was 3.3 x 10(-2) mg/L, in moderately resistant strains it was 1.3 x 10(-1) mg/L and in highly resistant strains it was 7.5 mg/L. No degradation of mupirocin could be detected during extended incubation of the antibiotic with cell free extracts from four resistant S. aureus strains. We conclude that the production of a modified IRS enzyme is the major cause of mupirocin resistance in the strains studied.

Homology of yeast mitochondrial leucyl-tRNA synthetase and isoleucyl- and methionyl-tRNA synthetases of Escherichia coli.

Respiratory deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G59 are pleiotropically deficient in respiratory chain components and in mitochondrial ATPase. This phenotype has been shown to be a consequence of mutations in a nuclear gene coding for mitochondrial leucyl-tRNA synthetase. The structural gene (MSL1) coding for the mitochondrial enzyme has been cloned by transformation of two different G59 mutants with genomic libraries of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein of 894 residues with a molecular weight of 101,936. The amino-terminal sequence (30-40 residues) has a large percentage of basic and hydroxylated residues suggestive of a mitochondrial import signal. The cloned MSL1 gene was used to construct a strain in which 1 kb of the coding sequence was deleted and substituted with the yeast LEU2 gene. Mitochondrial extracts obtained from the mutant carrying the disrupted MSL1::LEU2 allele did not catalyze acylation of mitochondrial leucyl-tRNA even though other tRNAs were normally charged. These results confirmed the correct identification of MSL1 as the structural gene for mitochondrial leucyl-tRNA synthetase. Mutations in MSL1 affect the ability of yeast to grow on nonfermentable substrates but are not lethal indicating that the cytoplasmic leucyl-tRNA synthetase is encoded by a different gene. The primary sequence of yeast mitochondrial leucyl-tRNA synthetase has been compared to other bacterial and eukaryotic synthetases. Significant homology has been found between the yeast enzyme and the methionyl- and isoleucyl-tRNA synthetases of Escherichia coli. The most striking primary sequence homology occurs in the amino-terminal regions of the three proteins encompassing some 150 residues. Several smaller domains in the more internal regions of the polypeptide chains, however, also exhibit homology. These observations have been interpreted to indicate that the three synthetases may represent a related subset of enzymes originating from a common ancestral gene.

Structure of the yeast isoleucyl-tRNA synthetase gene (ILS1). DNA-sequence, amino-acid sequence of proteolytic peptides of the enzyme and comparison of the structure to those of other known aminoacyl-tRNA synthetases.

The ILS1 gene encoding for cytoplasmic isoleucyl-tRNA synthetase from Saccharomyces cerevisiae was subcloned from a 5.4-kb insert of the shuttle vector YEp13 to M13mp8 and M13mp9. Nucleotide sequence analysis of a 4.3-kb BamHI-HpaI fragment revealed a single open reading frame from which we deduced the amino-acid sequence of the enzyme. Independently obtained amino-acid sequence information from ten tryptic peptides of the purified enzyme confirmed the gene-derived structure. The enzyme is comprised of 1073 amino-acids consistent with earlier determinations of its molecular mass. The codon usage of ILS1 is typical of abundant yeast proteins. A significant homology to E. coli isoleucyl- and valyl-tRNA synthetases as well as to yeast valyl-tRNA synthetase was detected. The characteristic amino-acid residues of the aminoacyl-adenylate site and of the potential binding site of the 3'-end of tRNA found in other synthetases are present in the structure.