Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase.

H Zalkin,J L Paluh,M Van Cleemput,W S Moye,C Yanofsky

doi:10.1016/s0021-9258(17)43193-0

Abstract

Saccharomyces cerevisiae anthranilate synthase:indole-3-glycerol phosphate synthase is a multifunctional hetero-oligomeric enzyme encoded by genes TRP2 and TRP3. TRP2, encoding anthranilate synthase Component I, was cloned by complementation of a yeast trp2 mutant. The nucleotide sequence of TRP2 as well as that of TRP3 were determined. The deduced anthranilate synthase Component I primary structure from yeast exhibits only limited similarity to that of the corresponding Escherichia coli subunit encoded by trpE. On the other hand, yeast anthranilate synthase Component II and indole-3-glycerol phosphate synthase amino acid sequences from TRP3 are clearly homologous with the corresponding sequences of the E. coli trpG and trpC polypeptide segments and thereby establish the bifunctional structure of TRP3 protein. Based on comparisons of TRP3 amino acid sequence with homologous sequences from E. coli and Neurospora crassa, an 11-amino acid residue connecting segment was identified which fuses the trpG and trpC functions of the bifunctional TRP3 protein chain. These comparisons support the conclusion that the amino acid sequence of connectors in homologous multifunctional enzymes need not be conserved. Connector function is thus not dependent on a specific sequence. Nuclease S1 mapping was used to identify mRNA 5' termini. Heterogeneous 5' termini were found for both TRP2 and TRP3 mRNA. TRP2 and TRP3 5'-flanking regions were analyzed for sequences that might function in regulation of these genes by the S. cerevisiae general amino acid control system. The 9 base pair direct repeat (Hinnebusch, A.G., and Fink, G.R. (1983) J. Biol. Chem. 258, 5238-5247) and inverted repeats were identified in the 5'-flanking sequences of TRP2 and TRP3.

Highlights

From the DeDartment of Biochemistry, Purdue University, West Lafayette, India4n7a907 and §the Departmentof Biological Sciences, Stdnford Uniiersity, Stanford, California 94305
AS Icatalyzes anNH3-dependent functional hetero-oligomeric enzymeencoded by genes synthesis of anthranilate fromchorismate.AS I1 forms a TRPB and TRP3
Cloning of Yeast TRP2-TRP2 was isolated from a yeast w m plasmid pool containing BamHI fragments ligated into the h f 1

Summary

RESULTS

This sequenceencodesa TRP3proteinchain of 484 amino acids having a calculated M , of 53,523. In E. coli, trpGenzymecomprises approximately 195residues at the NHz-terminal endof a bifunctional AS II/anthranilate phosphoribosyltransferase protein chain,while trpC protein comprisesanNH2-terminal segment of an indole-3-glycerol-P synthase phosphoribosylanthranilate isomerase bifunctional enzyme In both segmentsof TRP3 enzyme, blocks of amino acid residues identical withE. The longest segment of TRP3 lacking sequence homology with the E. coli enzymes is 34 amino acidsbetweenresidues 191-224 This region contains the aminoacids which connect trpG and trpC functions in TRP3.

HY ASAISVLTDEKYJQGsFNFLPIISQLAPQPILCZ

DISCUSSION

Of which exhibit general likewise have

John Wiley