Arabidopsis thaliana tryptophan synthase alpha: gene cloning, expression, and subunit interaction.

  • Radwanski E
  • Zhao J
  • Last R
  • 1

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Abstract

The tryptophan synthase alpha subunit catalyzes the conversion of indole-3-glycerolphosphate to indole, the penultimate reaction in the biosynthesis of the essential amino acid tryptophan. A cDNA encoding Arabidopsis thaliana tryptophan synthase alpha(TSA1) was isolated by complementation of an Escherichia coli delta trpA mutation and by polymerase chain reaction amplification from a cDNA library using degenerate primers. A TSA1 genomic clone was also isolated and 5 kb of the DNA sequence determined. A single sequence in the Arabidopsis genome with homology to the TSA1 cDNA was detected by high-stringency genomic Southern blot hybridization. In contrast under hybridization conditions of reduced stringency, one or two additional homologous sequences were observed. A 1.4 kb transcript was detected in wild-type RNA with the TSA1 cDNA as a probe. Several lines of evidence, including immunoaffinity chromatography, suggest that the active A. thaliana tryptophan synthase enzyme consists of a heterosubunit complex, presumably analogous to the prokaryotic alpha 2 beta 2 complex. Immunoblot analysis indicated that the plant alpha and beta subunits are present throughout development.

Author-supplied keywords

  • Affinity
  • Amino Acid Sequence
  • Arabidopsis
  • Arabidopsis: enzymology
  • Arabidopsis: genetics
  • Base Sequence
  • Blotting
  • Chromatography
  • Cloning
  • DNA Primers
  • DNA Primers: chemistry
  • Gene Expression Regulation
  • Genes
  • Genetic
  • Immunoblotting
  • Molecular
  • Molecular Sequence Data
  • Northern
  • Plant
  • Plant: genetics
  • Recombinant Proteins
  • Recombinant Proteins: genetics
  • Recombinant Proteins: isolation & purification
  • Sequence Alignment
  • Sequence Analysis
  • Southern
  • Transformation
  • Tryptophan Synthase
  • Tryptophan Synthase: chemistry
  • Tryptophan Synthase: genetics
  • Tryptophan Synthase: metabolism

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Authors

  • E R Radwanski

  • J Zhao

  • R L Last

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