Aspartyl-tRNA synthetase from Escherichia coli: cloning and characterisation of the gene, homologies of its translated amino acid sequence with asparaginyl- and lysyl-tRNA synthetases.
By screening of an Escherichia coli plasmidic library using antibodies against aspartyl-tRNA synthetase (AspRS) several clones were obtained containing aspS, the gene coding for AspRS. We report here the nucleotide sequence of aspS and the corresponding primary structure of the aspartyl-tRNA synthetase, a protein of 590 amino acid residues with a Mr 65,913, a value in close agreement with that observed for the purified protein. Primer extension analysis of the aspS mRNA using reverse transcriptase located its 5'-end at 94 nucleotides upstream of the translation initiation AUG; nuclease S1 analysis located the 3'-end at 126 nucleotides downstream of the stop codon UGA. Comparison of the DNA-derived protein sequence with known aminoacyl-tRNA sequences revealed important homologies with asparaginyl- and lysyl-tRNA synthetases from E.coli; more than 25% of their amino acid residues are identical, the homologies being distributed preferencially in the first part and the carboxy-terminal end of the molecule. Mutagenesis directed towards a consensus tetrapeptide (Gly-Leu-Asp-Arg) and the carboxy-terminal end showed that both domains could be implicated in catalysis as well as in ATP binding.
Eriani
G
G
Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France.
Dirheimer
G
G
Gangloff
J
J
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D001226:Aspartate-tRNA Ligase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008250:Lysine-tRNA Ligase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl; D015183:Restriction Mapping; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D003062:Codon; D012346:RNA, Transfer, Amino Acyl; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase; C029194:asparaginyl-tRNA synthetase; D008250:Lysine-tRNA Ligase