ARABIDOPSIS-THALIANA TRYPTOPHAN SYNTHASE-ALPHA - GENE CLONING, EXPRESSION, AND SUBUNIT INTERACTION

The tryptophan synthase a subunit catalyzes the conversion of indole-3 -glycerolphosphate to indole, the penultimate reaction in the biosynth esis of the essential amino acid tryptophan. A cDNA encoding Arabidops is thaliana tryptophan synthase alpha(TSA1) was isolated by complement ation of an Escherichia coli Delta trpA mutation and by polymerase cha in 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 a s a probe. Several lines of evidence, including immunoaffinity chromat ography, suggest that the active A. thaliana tryptophan synthase enzym e consists of a heterosubunit complex, presumably analogous to the pro karyotic alpha(2) beta(2) complex. Immunoblot analysis indicated that the plant a and beta subunits are present throughout development.