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Two mutations in the dispensable part of alanine tRNA synthetase which affect the catalytic activity

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Overview

authors

  • Jasin, M.
  • Regan, L.
  • Schimmel, Paul

publication date

  • 1985

journal

  • Journal of Biological Chemistry  Journal

abstract

  • 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.

subject areas

  • Acylation
  • Adenosine Monophosphate
  • Alanine
  • Alanine-tRNA Ligase
  • Amino Acyl-tRNA Synthetases
  • Base Sequence
  • Cloning, Molecular
  • Codon
  • Escherichia coli
  • Genes
  • Genes, Bacterial
  • Molecular Weight
  • Mutation
  • Plasmids
  • Transformation, Bacterial
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

PubMed ID

  • 3882689
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Additional Document Info

start page

  • 2226

end page

  • 2230

volume

  • 260

issue

  • 4

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