Abstract
Transcription of the gene for phosphoribosyl-anthranilate isomerase (TRP1) from the TRP1 promoter is initiated only approximately half as frequently as, for example, from the TRP3 promoter, but TRP1 mRNA is approximately twice as stable as TRP3 mRNA. Therefore, the steady state amount of TRP1 mRNA in yeast cells, grown without amino acid limitation, is similar to the steady-state amount of TRP3 mRNA. The protein concentration of both enzymes in yeast cells is about the same, but the basal specific enzyme activity in permeabilized cells of the TRP1 gene product N-(5'-phosphoribosyl-1)-anthranilate isomerase is about 2-3 times higher than that of any of the other TRP enzymes. According to the kinetic parameters of the purified isomerase protein, the enzyme is more active than, for example, the purified TRP3 enzyme indoleglycerol-phosphate synthase. It is suggested that the TRP1 gene of Saccharomyces cerevisiae might be the result of a rearrangement event, separating the N-(5'-phosphoribosyl-1)-anthranilate isomerase domain from the indoleglycerol-phosphate synthase domain and putting the catalytically more active isomerase domain behind a weak and nonregulated constitutive promoter.
Highlights
The suggested that theTRPl gene of Saccharomyces cere- TRPl gene is used in many yeast vectors asa selectable visiae might be the result of a rearrangement event, marker
The TRPl gene product PRA isomerase catalyzes a practically irreversible Amadori rearrangement, the third step in enzymes appear to bemore highly organized in eukaryotic tryptophan biosynthesis
If one compares the total amount(laneZ),the increase in mRNA correlated well with of mRNA of the chromosomal TRP3 gene in strain X2180- the derepression factors observed with the TRP3-lacZ fusion lA, expressed under nonderepressing conditions(laneI ), and (Table I ) and thegene product InGP synthase (Fig. 4).This served as a controlfor equivalent poly(A)+enrichment of both
Summary
The TRPl gene product PRA isomerase catalyzes a practically irreversible Amadori rearrangement, the third step in enzymes appear to bemore highly organized in eukaryotic tryptophan biosynthesis. Four genes encode the seven func- gene and its gene product PRA isomerase in the tryptophan tional domains of the tryptophan pathway One of these genes biosynthesis of S. cerevisiae.The expression of the TRPl gene codes for a trifunctional polypeptide, NH2-glutamine amido-. Phate synthase; PRA isomerase, N-(5’-phosphoribosyl-l)-anthranilate isomerase; CDRP, 1-(o-carboxypheny1amino)-1-deoxyribulose 5-phosphate;kb, kilobase; bp, base pair; PRPP, 5-phosphoribosyl 1pyrophosphate; X-Gal, 5-bromo-4-chloro-3-indolylP-D-galactoside; GAP, glyceraldehyde 3-phosphate; ONPG, o-nitrophenyl P-D-galaCtoside; SDS, sodium dodecyl sulfate; FPLC, fast protein liquid chromatography. The TRPl gene product PRA isomerase was purified, characterized, and compared to the purified TRP3 gene product InGP synthase (Prantl etal., 1985)
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