Coevolution of transcriptional and allosteric regulation at the chorismate metabolic branch point of Saccharomyces cerevisiae

Abstract

Control of transcription and enzyme activities are two interwoven regulatory systems essential for the function of a metabolic node. Saccharomyces cerevisiae strains differing in enzyme activities at the chorismate branch point of aromatic amino acid biosynthesis were constructed by recombinant DNA technology. Expression of an allosterically unregulated, constitutively activated chorismate mutase encoded by the ARO7(T226I) (ARO7(c)) allele depleted the chorismate pool. The resulting tryptophan limitation caused growth defects, which could be counteracted only by transcriptional induction of TRP2 encoding the competing enzyme anthranilate synthase. ARO7 expression is not transcriptionally regulated by amino acids. Transcriptional activation of the ARO7(c) allele led to stronger growth retardation upon tryptophan limitation. The same effect was achieved by removing the competing enzyme anthranilate synthase, which is encoded by the TRP2 gene, from the transcriptional control. The allelic situation of ARO7(c) being under general control instead of TRP2 resulted in severe growth defects when cells were starved for tryptophan. In conclusion, the specific regulatory pattern acting on enzymatic activities at the first metabolic node of aromatic amino acid biosynthesis is necessary to maintain proper flux distribution. Therefore, the evolution of the sophisticated allosteric regulation of yeast chorismate mutase requires as prerequisite (i) that the encoding ARO7 gene is not transcriptionally regulated, whereas (ii) the transcription of the competing feedback-regulated anthranilate synthase-encoding gene is controlled by availability of amino acids.

Figure 1

Interplay between transcriptional and enzyme regulation at the first branch point of aromatic amino acid biosynthesis in yeast. The metabolic node emerging from chorismate is schematically shown with the enzymatic activities chorismate mutase (CM) and anthranilate synthase (AAS) as black ovals. The encoding genomic loci are drawn as bars with transcriptional start sites indicated by broken arrows. Positive feedbacks and inductions are indicated by ⊕, feedback inhibition by ⊝.

Figure 2

Specific AAS (Upper) and CM (Lower) activities of S. cerevisiae strains with altered regulatory properties of the branch point enzymes. Activities were determined from cells cultivated in minimal medium expressing either no functional Gcn4p (−), low levels (+), or high levels (+++) of Gcn4p. The genotypes of all strains are indicated with ^pA∷ and ^pT∷ representing ARO7 and TRP2 promoter fusions, respectively. All values are the means of two independent measurements with a standard deviation not exceeding 20%.

Figure 3

Northern hybridization analyses of S. cerevisiae strains expressing ARO7^c and TRP2 with altered dependence on the general control activator Gcn4p. Cells were cultivated in minimal medium and expressed no functional Gcn4p (−) or low levels (+) or high levels (+++) of Gcn4p, as indicated. In each lane, 25 μg of total RNAs was hybridized successively with probes specific for ARO7/TRP2 and ACT1 (encoding actin). For quantification, steady-state transcript levels were standardized with respect to ACT1 levels and the ratio between ARO7^c and TRP2 transcript levels is indicated at the bottom. All values are the means of two independent measurements with a standard deviation not exceeding 20%.