RESUMEN
Multigene families are observed in all genomes sequenced so far and are the reflection of key evolutionary mechanisms. The DUP240 family, identified in Saccharomyces cerevisiae strain S288C, is composed of 10 paralogs: seven are organized as two tandem repeats and three are solo ORFs. To investigate the evolution of the three solo paralogs, YAR023c, YCR007c and YHL044w, we performed a comparative analysis between 15 S.cerevisiae strains. These three ORFs are present in all strains and the conservation of synteny indicates that they are not frequently involved in chromosomal reshaping, in contrast to the DUP240 ORFs organized in tandem repeats. Our analysis of nucleotide and amino acid variations indicates that YAR023c and YHL044w fix mutations more easily than YCR007c, although they all belong to the same multigene family. This comparative analysis was also conducted with five arbitrarily chosen Ascomycetes-specific genes and five arbitrarily chosen common genes (genes that have a homolog in at least one non-Ascomycetes organism). Ascomycetes-specific genes appear to be diverging faster than common genes in the S.cerevisiae species, a situation that was previously described between different yeast species. Our results point to the strong contribution, during DNA sequence evolution, of allelic recombination besides nucleotide substitution.
Asunto(s)
Evolución Molecular , Familia de Multigenes/genética , Filogenia , Recombinación Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Mapeo Cromosómico , ADN de Hongos/química , ADN de Hongos/genética , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
The yeast Saccharomyces cerevisiae possesses two methionyl-tRNA synthetases (MetRS), one in the cytoplasm and the other in mitochondria. The cytoplasmic MetRS has a zinc-finger motif of the type Cys-X(2)-Cys-X(9)-Cys-X(2)-Cys in an insertion domain that divides the nucleotide-binding fold into two halves, whereas no such motif is present in the mitochondrial MetRS. Here, we show that tightly bound zinc atom is present in the cytoplasmic MetRS but not in the mitochondrial MetRS. To test whether the presence of a zinc-binding site is required for cytoplasmic functions of MetRS, we constructed a yeast strain in which cytoplasmic MetRS gene was inactivated and the mitochondrial MetRS gene was expressed in the cytoplasm. Provided that methionine-accepting tRNA is overexpressed, this strain was viable, indicating that mitochondrial MetRS was able to aminoacylate tRNA(Met) in the cytoplasm. Site-directed mutagenesis demonstrated that the zinc domain was required for the stability and consequently for the activity of cytoplasmic MetRS. Mitochondrial MetRS, like cytoplasmic MetRS, supported homocysteine editing in vivo in the yeast cytoplasm. Both MetRSs catalyzed homocysteine editing and aminoacylation of coenzyme A in vitro. Thus, identical synthetic and editing functions can be carried out in different structural frameworks of cytoplasmic and mitochondrial MetRSs.
Asunto(s)
Citoplasma/enzimología , Metionina-ARNt Ligasa/química , Metionina-ARNt Ligasa/metabolismo , Mitocondrias/enzimología , Saccharomyces cerevisiae/enzimología , Acilación , Secuencia de Aminoácidos , Sitios de Unión , Coenzima A/metabolismo , Cisteína/genética , Cisteína/metabolismo , Genes Fúngicos/genética , Prueba de Complementación Genética , Homocisteína/genética , Homocisteína/metabolismo , Cinética , Metionina/metabolismo , Metionina-ARNt Ligasa/genética , Datos de Secuencia Molecular , Mutación/genética , Transporte de Proteínas , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Relación Estructura-Actividad , Zinc/metabolismo , Dedos de Zinc/genética , Dedos de Zinc/fisiologíaRESUMEN
Previous work suggested that the presence of the anticodon CAU alone was enough to confer methionine acceptance to a tRNA. Conversions of Escherichia coli nonmethionine tRNAs to a methionine-accepting species were obtained by substitutions reconstructing the whole methionine anticodon loop together with preservation (or introduction) of the acceptor stem base A73. We show here that the CAU triplet alone is unable to confer methionine acceptance when transplanted into a yeast aspartic tRNA. Both non-anticodon bases of the anticodon loop of yeast tRNA(Met) and A73 are required in addition to CAU for methionine acceptance. The importance of these non-anticodon bases in other CAU-containing tRNA frameworks was also established. These specific non-anticodon base interactions make a substantial thermodynamic contribution to the methionine acceptance of a transfer RNA.
Asunto(s)
Anticodón/metabolismo , Metionina/metabolismo , ARN de Transferencia de Metionina/metabolismo , Saccharomyces cerevisiae/genética , Anticodón/genética , Secuencia de Bases , Cinética , Modelos Estructurales , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN de Transferencia de Metionina/genética , Transcripción GenéticaRESUMEN
As for Escherichia coli methionine tRNAs, the anticodon triplet of yeast tRNA(Met) plays an important role in the recognition by the yeast methionyl-tRNA synthetase (MetRS), indicating that this determinant for methionine identity is conserved in yeast. Efficient aminoacylation of the E. coli tRNA(Met) transcript by the heterologous yeast methionine enzyme also suggests conservation of the protein determinants that interact with the CAU anticodon sequence. We have analysed by site-directed mutagenesis the peptide region 655 to 663 of the yeast MetRS that is equivalent to the anticodon binding region of the E. coli methionine enzyme. Only one change, converting Leu658 into Ala significantly reduced tRNA aminoacylation. Semi-conservative substitutions of L658 allow a correlation to be drawn between side-chain volume of the hydrophobic residue at this site and activity. The analysis of the L658A mutant shows that Km is mainly affected. This suggests that the peptide region 655 to 663 contributes partially to the binding of the anticodon, since separate mutational analysis of the anticodon bases shows that kcat is the most critical parameter in the recognition of tRNA(Met) by the yeast synthetase. We have analysed the role of peptide region (583-GNLVNR-588) that is spatially close to the region 655 to 663. Replacements of residues N584 and R588 reduces significantly the kcat of aminoacylation. The peptide region 583-GNLVNR-588 is highly conserved in all MetRS so far sequenced. We therefore propose that the hydrogen donor/acceptor amino acid residues within this region are the most critical protein determinants for the positive selection of the methionine tRNAs.
Asunto(s)
Anticodón/metabolismo , Metionina-ARNt Ligasa/metabolismo , ARN de Transferencia de Metionina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Relación Estructura-ActividadRESUMEN
Sequence comparisons among methionyl-tRNA synthetases from different organisms reveal only one block of homology beyond the last beta strand of the mononucleotide fold. We have introduced a series of semi-conservative amino acid replacements in the conserved motif of yeast methionyl-tRNA synthetase. The results indicate that replacements of two polar residues (Asn584 and Arg588) affected specifically the aminoacylation reaction. The location of these residues in the tertiary structure of the enzyme is compatible with a direct interaction of the amino acid side-chains with the tRNA anticodon.
Asunto(s)
Anticodón , Metionina-ARNt Ligasa/genética , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Escherichia coli/genética , Cinética , Metionina-ARNt Ligasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Plásmidos , Conformación Proteica , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Yeast methionyl-tRNA synthetase has a long N-terminal extension fused to the mononucleotide binding fold that occurs at the N-terminal end of the homologous E coli enzyme. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA amino-acylation steps than on the adenylate formation step. The Km for ATP and methionine are unaltered, indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3, which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the 3-D structure of the homologous E coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration of the catalytic residues. The tRNA(Met) anticodon binding region of the E coli enzyme has recently been characterized. By mutagenesis of the topologically equivalent region in the yeast enzyme, we could identify residues that alter specifically the aminoacylation of the tRNA. Leu 658 provides a van der Waals contact that is critical for the recognition of the yeast tRNA.