Identification of YHR019 in Saccharomyces cerevisiae chromosome VIII as the gene for the cytosolic asparaginyl-tRNA synthetase.
Exploiting the asparagine auxotrophy of the Saccharomyces cerevisiae mutant strain 8556a, we have isolated the gene for the cytosolic asparaginyl-tRNA synthetase (AsnRS) of S. cerevisiae, by functional complementation of the mutation affecting this strain. The isolated gene could be identified to the open reading frame YHR019, called DED81, located on chromosome VIII. The mutant gene from the 8556a strain, asnrs-1, was amplified from genomic DNA by PCR. This gene contains a point mutation, leading to the replacement of a glycine residue by a serine in a region of the protein probably important for the asparaginyl-adenylate recognition. The protein encoded by YHR019 is very similar to cytosolic AsnRS from other eukaryotic sources. In a phylogenetic analysis based on AsnRS sequences from various organisms, the eukaryotic sequences were clustered. Expression of YHR019 in Escherichia coli demonstrated that a yeast AsnRS activity was produced. The recombinant enzyme was purified to homogeneity in three chromatography steps. We showed that the recombinant S. cerevisiae AsnRS was able to charge unfractionated yeast tRNA, but not E. coli tRNA, with asparagine.
Landrieu
I
I
Unité de Microbiologie, Faculté Universitaire des Sciences Agronomiques de Gembloux, Belgium. microbio@fsagx.ac.be
Vandenbol
M
M
Leberman
R
R
Portetelle
D
D
Hartlein
M
M
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001226:Aspartate-tRNA Ligase; D003600:Cytosol / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005784:Gene Amplification; D005800:Genes, Fungal; D010802:Phylogeny; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D012346:RNA, Transfer, Amino Acyl; D011994:Recombinant Proteins; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase; C029194:asparaginyl-tRNA synthetase