Regulation of activity and synthesis of N-acetylglutamate synthase from Saccharomyces cerevisiae.

Feedback inhibition of N-acetylgutamate synthase in a particulate fraction from Saccharomyces cerevisiae by L-arginine was synergistically enhanced by N-actylglutamate, whereas coenzyme A let to an additive enhancement of arginine inhibition. N-acetylglutamate synthase was not inhibited by polyamines, nor was the enzyme inactivated by incubation in the presence of coenzyme A and zinc ions. Evidence was obtained for the involvement of at least three different regulatory mechanisms in the expression of N-acetylglutamate synthase: arginine-specific repression, glucose repression and general amino acid control. The combined action of these control mechanisms led to a 90-fold variation in the specific activity of the enzyme.     

Dual regulation of the synthesis of the arginine pathway carbamoylphosphate synthase of Saccharomyces cerevisiae by specific and general controls of amino acid biosynthesis.

Summary The synthesis of the arginine pathway carbamoylphosphate synthase (CPSase A) of Saccharomyces cerevisiae is subject to two control mechanisms. One mechanism is specific for CPSase A and is exerted by arginine; it probably involves a repressoroperator type of interaction. This specific mechanism regulates the expression of gene cpaI coding for the small glutaminase subunit of CPSase A but has little influence on the production of the large subunit of the enzyme, a product of gene cpaII. This large component, which alone has no biological significance, accumulates freely under conditions of arginine repression. The second mechanism is general: it controls enzyme synthesis in a number of amino acid biosynthetic pathways in addition to the arginine sequence. Two types of evidence that this general mechanism participates in the control of CPSase A synthesis are presented: (1) Derepression upon starvation for any amino acid of which the synthesis is subject to this general control; and (2) repression during growth in amino acid-rich medium. In contrast to the specific mechanism, the general mechanism regulates the expression of both the cpaI and cpaII genes.     

Biological role of the general control of amino acid biosynthesis in Saccharomyces cerevisiae.

The biological role of the "general control of amino acid biosynthesis" has been investigated by analyzing growth and enzyme levels in wild-type, bradytrophic, and nonderepressing mutant strains of Saccharomyces cerevisiae. Amino acid limitation was achieved by using either bradytrophic mutations or external amino acid imbalance. In the wild-type strain noncoordinate derepression of enzymes subject to the general control has been found. Derepressing factors were in the order of 2 to 4 in bradytrophic mutant strains grown under limiting conditions and only in the order of 1.5 to 2 under the influence of external amino acid imbalance. Nonderepressing mutations led to slower growth rates under conditions of amino acid limitation, and no derepression of enzymes under the general control was observed. The amino acid pools were found to be very similar in the wild type and in nonderepressing mutant strains under all conditions tested. Our results indicate that the general control affects all branched amino acid biosynthetic pathways, namely, those of the aromatic amino acids and the aspartate family, the pathways for the basic amino acids lysine, histidine, and arginine, and also the pathways of serine and valine biosyntheses.     

A segment of mRNA encoding the leader peptide of the CPA1 gene confers repression by arginine on a heterologous yeast gene transcript.

The expression of the yeast gene CPA1, which encodes the small subunit of the arginine pathway carbamoylphosphate synthetase, is repressed by arginine at a translational level. CPA1 mRNA contains a 250-nucleotide-long leader which includes a 25-codon upstream open reading frame (uORF). Oligonucleotide site-directed mutagenesis of this uORF as well as sequencing of constitutive cis-dominant mutations has suggested that the leader peptide product of the CPA1 uORF is an essential negative element for repression of the CPA1 gene by arginine. In this work, a series of deletions affecting the regions 5' and 3' to the uORF in the leader sequence was constructed. The arginine-dependent repression of CPA1 was little affected in these constructions, indicating that these regions are not essential for the regulatory response. This conclusion was further supported by the finding that inserting the mRNA segment encoding the leader peptide sequence of CPA1 in the leader sequence of another gene, namely, GCN4, places this gene under arginine repression. Similarly, the behavior of fusions of the leader sequence of CPA1 with those of ARG4 or GAL10 confirmed that the regions of this leader located upstream and downstream from the uORF are dispensable for the regulation by arginine. Finally, a set of substitution mutations which modify the uORF nucleotide sequence while leaving unchanged the corresponding amino acid sequence was constructed. The mutations did not affect the repression of CPA1 by arginine. The data presented in this paper consequently agree with the conclusion that the leader peptide itself is the main element required for the translational repression of CPA1.     

Evolutionary changes in the fungal carbamoyl-phosphate synthetase small subunit gene and its associated upstream open reading frame

The Neurospora crassa arg-2 and the Saccharomyces cerevisiae ortholog CPA1 encode the arginine-specific carbamoyl-phosphate synthetase (CPS-A) small subunit. Arginine decreases synthesis of this subunit through the action of a 5' upstream open reading frame in the mRNA that encodes a cis-regulatory element, the arginine attenuator peptide (AAP), which stalls ribosomes in response to arginine. We performed a comparative analysis of the genomic structure and predicted peptide sequence of the AAP and CPS-A small subunit across many fungi. Differences at the genomic level included variation in intron number and position within the AAP and CPS-A coding regions and differences in known regulatory motifs. Although differences exist in AAP sequence, there were three absolutely conserved amino acid residues in the predicted peptide, including an aspartic acid crucial for arginine-dependent regulation of arg-2 and CPA1. A diverged Basidiomycete AAP was shown to retain function as an Arg-specific negative regulator of translation.