Two mutations in the dispensable part of alanine tRNA synthetase which affect the catalytic activity.
Two previously described chromosomal mutant alleles, alaS4 and alaS5, of Escherichia coli Ala-tRNA synthetase have been analyzed. Each causes a sharp diminution in aminoacylation activity and disrupts the alpha 4 tetramer structure of identical chains of 875 amino acids; neither mutation significantly disturbs the activity for synthesis of alanyladenylate. The location of each mutation within the structural gene has been mapped by marker rescue with specific gene fragments. Each mutant allele was cloned from the genome by reciprocal recombination with a multicopy plasmid that contains segments of alaS which flank the respective mutations. Further analysis established: 1) a single G----A transition results in a Gly----Asp change for each mutant allele at codon 674 (alaS4) and at codon 677 (alaS5). 2) The mutations are in the oligomerization domain, about 200 amino acids beyond the C-terminal side of the catalytic domain that previously was mapped by deletion analysis; the mutations are, thus, in a part of the polypeptide which is dispensable for catalytic activity. 3) For both mutant enzymes, there is little effect of the mutation on the Km for tRNAAla; kcat for aminoacylation is decreased by an order of magnitude. These point mutations reveal a subtle integration of the catalytic core with parts of the polypeptide that are not essential for catalytic activity.
D000215:Acylation; D000249:Adenosine Monophosphate / Q000096:biosynthesis; D000409:Alanine / Q000378:metabolism; D000412:Alanine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D014169:Transformation, Bacterial
D003062:Codon; D000249:Adenosine Monophosphate; D000604:Amino Acyl-tRNA Synthetases; D000412:Alanine-tRNA Ligase; D000409:Alanine