In vitro studies of the binding of the ARGR proteins to the ARG5,6 promoter.

ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae. We show here that they are also required in vitro to observe a protein-DNA complex with the promoter of the ARG5,6 gene. The specific binding of ARGR proteins in vitro is stimulated by arginine. Antibodies raised against a synthetic MCM1 polypeptide retard the migration of ARGR-DNA complex on gel mobility shift assays. This result suggests that MCM1 could be an additional regulatory element of arginine metabolism.

Genetic evidence for a role for MCM1 in the regulation of arginine metabolism in Saccharomyces cerevisiae.

ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae. We have shown that MCM1 is part of the ARGR regulatory complex, by in vitro binding experiments, at the ARGR5,6 promoter. The participation of MCM1 in the regulation of arginine metabolism is confirmed by the behavior of an mcm1-gcn4 mutant, which is affected in the repression of arginine anabolic genes. In this mcm1 mutant, synthesis of the catabolic enzymes is rather constitutive, but this derepression requires the integrity of the ARGR system and of the target sequences of these proteins in the CAR1 promoter. Our in vitro binding experiments confirm the presence of MCM1 in the protein complex interacting with the promoters of the catabolic CAR1 and CAR2 genes. This is the first in vivo transcription role ascribed to MCM1 other than its role in the transcription of cell-type-specific genes.

Determination of the DNA-binding sequences of ARGR proteins to arginine anabolic and catabolic promoters.

ARGRI, ARGRII, and ARGRIII proteins regulate the expression of arginine anabolic and catabolic genes. The integrity of these three proteins is required to observe the formation of a DNA-protein complex with the different promoters of arginine coregulated genes. A study of deletions and point mutations created in the 5' noncoding region of ARG3, ARG5,6, CAR1, and CAR2 genes shows that at least two regions, called BoxA and BoxB, are required for proper regulation of these genes by arginine and ARGR proteins. By gel retardation assay and DNase I footprinting analysis, we have determined precisely the target of the ARGR proteins. Sequences in and around BoxA are necessary for ARGR binding to these four promoters in vitro, whereas sequences in and around BoxB are clearly protected against DNase I digestion only for CAR1. Sequences present at BoxA and BoxB are well conserved among the four promoters. Moreover, pairing can occur between sequences at BoxA and BoxB which could lead to the creation of secondary structures in ARG3, ARG5,6, CAR1, and CAR2 promoters, favoring the binding of ARGR proteins in vivo.

Dissection of the bifunctional ARGRII protein involved in the regulation of arginine anabolic and catabolic pathways.

ARGRII is a regulatory protein which regulates the arginine anabolic and catabolic pathways in combination with ARGRI and ARGRIII. We have investigated, by deletion analysis and fusion to LexA protein, the different domains of ARGRII protein. In contrast to other yeast regulatory proteins, 92% of ARGRII is necessary for its anabolic repression function and 80% is necessary for its catabolic activator function. We can define three domains in this protein: a putative DNA-binding domain containing a zinc finger motif, a region more involved in the repression activity located around the RNase-like sequence, and a large activation domain.

ArgRII, a component of the ArgR-Mcm1 complex involved in the control of arginine metabolism in Saccharomyces cerevisiae, is the sensor of arginine.

Repression of arginine anabolic genes and induction of arginine catabolic genes are mediated by a three-component protein complex, interacting with specific DNA sequences in the presence of arginine. Although ArgRI and Mcm1, two MADS-box proteins, and ArgRII, a zinc cluster protein, contain putative DNA binding domains, alone they are unable to bind the arginine boxes in vitro. Using purified glutathione S-transferase fusion proteins, we demonstrate that ArgRI and ArgRII1-180 or Mcm1 and ArgRII1-180 are able to reconstitute an arginine-dependent binding activity in mobility shift analysis. Binding efficiency is enhanced when the three recombinant proteins are present simultaneously. At physiological concentration, the full-length ArgRII is required to fulfill its functions; however, when ArgRII is overexpressed, the first 180 amino acids are sufficient to interact with ArgRI, Mcm1, and arginine, leading to the formation of an ArgR-Mcm1-DNA complex. Several lines of evidence indicate that ArgRII is the sensor of the effector arginine and that the binding site of arginine would be the region downstream from the zinc cluster, sharing some identity with the arginine binding domain of bacterial arginine repressors.

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.

General amino acid control and specific arginine repression in Saccharomyces cerevisiae: physical study of the bifunctional regulatory region of the ARG3 gene.

To characterize further the regulatory mechanism modulating the expression of the Saccharomyces cerevisiae ARG3 gene, i.e., the specific repression by arginine and the general amino acid control, we analyzed by deletion the region upstream of that gene, determined the nucleotide sequence of operator-constitutive-like mutations affecting the specific regulation, and examined the behavior of an ARG3-galK fusion engineered at the initiating codon of ARG3. Similarly to what was observed in previous studies on the HIS3 and HIS4 genes, our data show that the general regulation acts as a positive control and that a sequence containing the nucleotide TGACTC, between positions -364 and -282 upstream of the transcription start, functions as a regulatory target site. This sequence contains the most proximal of the two TGACTC boxes identified in front of ARG3. While the general control appears to modulate transcription efficiency, the specific repression by arginine displays a posttranscriptional component (F. Messenguy and E. Dubois, Mol. Gen. Genet. 189:148-156, 1983). Our deletion and gene fusion analyses confirm that the specific and general controls operate independently of each other and assign the site responsible for arginine-specific repression to between positions -170 and +22. In keeping with this assignment, the two operator-constitutive-like mutations were localized at positions -80 and -46, respectively, and thus in a region which is not transcribed. We discuss a hypothesis accounting for the involvement of untranscribed DNA in a posttranscriptional control.

Temperature-sensitive nonsense suppressors in yeast.

A single mutation leads to temperature-sensitive amber and ochre suppression in yeast. This mutation maps in or near the SUP4 gene on chromosome X.

Regulation of arginine metabolism in Saccharomyces cerevisiae: expression of the three ARGR regulatory genes and cellular localization of their products.

Three regulatory proteins are involved in the control of arginine metabolism in yeast: ARGRI, ARGRII and ARGRIII. The control region and part of the coding sequence of the ARGR genes were fused to the Escherichia coli lacZ gene. These chimeras were used to study the expression of the regulatory genes as well as the cellular compartmentalization of the regulatory products. Our results show that the three ARGR proteins are localized in the nucleus and that their synthesis is not regulated by arginine nor by any of the other ARGR products. However, some data suggest that the ARGRIII protein could control ARGRI activity.

Characterization of two new genes essential for vegetative growth in Saccharomyces cerevisiae: nucleotide sequence determination and chromosome mapping.

Based on nucleotide sequence determination, we have identified two new yeast genes FUN80 and FUN81 located on chromosome XIII. They are both essential for cellular growth but their function is still unknown. FUN80 is closely linked to the ARGRI (or ARG80) gene while FUN81 is located next to the ARGRII (or ARG81) gene. Interestingly, the proteins encoded by these two genes have a long stretch of acidic amino acids within their C-terminal portions.

Inositol polyphosphate kinase activity of Arg82/ArgRIII is not required for the regulation of the arginine metabolism in yeast.

Arg82, a nuclear regulator of diverse cellular processes in yeast, is an inositol polyphosphate kinase. Some defects such as the regulation of arginine metabolism observed in an arg82Delta, result from a lack of Mcm1 and Arg80 stability. We show here that neither the kinase activity of Arg82 nor inositol phosphates are required for the control of arginine metabolism. Arg82 mutations keeping kinase active affect the expression of arginine genes, whereas mutations in the kinase domain do not impair this metabolic control.

Concerted repression of the synthesis of the arginine biosynthetic enzymes by aminoacids: a comparison between the regulatory mechanisms controlling aminoacid biosyntheses in bacteria and in yeast.

It has been shown that in bacteria, besides specific regulatory mechanisms, the synthesis of aminoacid biosynthetic enzymes is also controlled by the endogenous aminoacid pool. The latter regulates the intracellular level of ppGpp, a positive effector of RNA messenger transcription. A similar regulatory control exists in yeast but does not appear to involve the same general effector. This was established by the observation that derepression of the enzymes belonging to several aminoacid biosynthetic pathways follows aminoacid starvation or tRNA discharging. We now report the repression of the arginine pathway by the total aminoacid pool. New mutations affecting the repressibility of the arginine enzymes as well as enzymes belonging to other aminoacid biosyntheses, when cells are grown in the presence of an excess of aminoacids, were identified.

Multiplicity of regulatory mechanisms controlling amino acid biosynthesis in Saccharomyces cerevisiae.

Transcriptional, post-transcriptional and translational regulatory mechanisms control gene expression of amino acid biosynthetic pathways in yeast. All three mechanisms are involved in the control of arginine metabolism.

Regulation of arginine biosynthesis in Saccharomyces cerevisiae: isolation of a cis-dominant, constitutive mutant for ornithine carbamoyltransferase synthesis.

A cis-dominant mutation linked to argF, the structural gene specifying ornithine carbamoyltransferase, and affecting the control of the synthesis of this enzyme has been obtained. The level of ornithine carbamoyltransferase in this mutation is depressed and less repressible by addition of L-arginine than it is in the wild-type strain. Of 38 tetrads analyzed, resulting from a cross of a strain harboring this mutation with a strain carrying an argF- mutation, none was a tetratype or a nonparental ditype. This operator mutation helps to define a negative mode of control of the synthesis of the arginine biosynthetic enzymes, as had been suggested earlier upon the isolation of argRI- (arg80), argRII- (arg81), and argRIII- (arg82) specific regulatory mutations.

The yeast ARGRII regulatory protein has homology with various RNases and DNA binding proteins.

Three regulatory proteins are involved in the post-transcriptional control of arginine metabolism in Saccharomyces cerevisiae: ARGRI, ARGRII and ARGRIII. The 880 amino acid ARGRII protein, like some DNA binding proteins, possesses in its N-terminal sequence a cysteine-rich region that presents homology to the zinc binding region of Escherichia coli aspartate transcarbamylase. ARGRII also has a region of 90 amino acids that is 30% homologous to the E. coli ARGR repressor. Moreover a 87 amino acid long sequence of ARGRII contains three stretches with significant homology to some viral, bacterial and pancreatic RNases. We propose a model in which the RNase-like sequence could regulate the expression of arginine anabolic messenger RNAs.

Integration of the multiple controls regulating the expression of the arginase gene CAR1 of Saccharomyces cerevisiae in response to differentnitrogen signals: role of Gln3p, ArgRp-Mcm1p, and Ume6p.

Expression of the catabolic gene encoding arginase in Saccharomyces cerevisiae, CAR1, is controlled by multiple nitrogen signals, such as the presence of the inducer, arginine, and the nature and amount of the nitrogen source. The present study has determined or confirmed the identity of the proteins involved in these different controls, as well as their targets in the CAR1 promoter. We show that Gln3p activates CAR1 expression through the GATAA sequences in the absence of an optimal nitrogen source, such as ammonia, glutamine or asparagine. Ume6p, which also controls the expression of early meiotic genes, represses CAR1 expression through a sequence called URS, as a function of nitrogen availability. Thus, the responses to the quality of the nitrogen source and to nitrogen starvation are achieved through different cis- and trans-regulatory elements. At least one of the multiple Rap1p and Abf1p binding sites is required for the basal transcription of the gene. The UAS(arg), containing the previously defined "arginine boxes" is the region that responds to the inducer through the action of the ArgRp-Mcm1p proteins, and its deletion alone significantly affects growth on arginine as sole nitrogen source. The functional UAS(arg) is about 60 nucleotides long, and contains two sequences homologous to the binding site for MADS-box proteins, to which ArgRIp and Mcm1p belong. No obvious palindromic sequence similar to the binding site of Gal4p, Ppr1p or Put3p is present in the UAS(arg), although ArgRIIp contains a Zn(II)2Cys6 motif. Interestingly, we have found that induction of CAR1 expression by arginine in the presence of an optimal nitrogen source is counteracted by Gln3p, independently of its action at the GATAA sequences.

Induction of "General Control" and thermotolerance in cdc mutants of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae starvation for a single amino acid activates the transcription of a set of genes belonging to different amino acid biosynthetic pathways (General Control, GC). We show that mutants affected in GC regulation are also affected in their response to thermal stress. Moreover, growth conditions that are known to induce heat shock proteins induce the GC response. However, unlike heat shock proteins, the transcriptional activator of GC, GCN4, is not induced after a short exposure to heat, and in gcn mutant strains induction of heat resistance is normal.

Isolation and characterization of the yeast ARGRII gene involved in regulating both anabolism and catabolism of arginine.

ARGRII is one of the three regulatory genes controlling arginine metabolism in yeast. From a pool of hybrid plasmids carrying Sau3A fragments representing the entire yeast genome, a DNA fragment containing the regulatory gene ARGRII was cloned by complementation of an argRII- mutation, which prevents growth on ornithine as sole nitrogen source. Cells containing the cloned DNA regained the ability to repress the synthesis of anabolic enzymes and to induce the synthesis of the catabolic ones, when arginine is present. The 6.2 kb cloned DNA fragment encodes five transcripts (2.8 kb, 1.3 kb, 0.75 kb, 0.45 kb, 0.45 kb), which were located by S1 endonuclease mapping. By marker rescue the argRII- mutations were mapped in the DNA region coding for the 2.8 kb transcript, showing its importance in the control mechanism. Subcloning experiments confirm this result. However, at present the role of the 0.75 kb and 1.3 kb transcripts in the ARGR+ phenotype is unclear.

Pleiotropic function of ArgRIIIp (Arg82p), one of the regulators of arginine metabolism in Saccharomyces cerevisiae. Role in expression of cell-type-specific genes.

ArgRIIIp (Arg82p), together with ArgRIp (Arg80p), ArgRIIp (Arg81p) and Mcm1p, regulates the expression of arginine anabolic and catabolic genes. An argRIII mutant constitutively expresses five anabolic enzymes and is impaired in the induction of the synthesis of two catabolic enzymes. A genomic disruption of the ARGRIII gene not only leads to an argR phenotype, but also prevents cell growth at 37 degrees C. The disrupted strain is sterile especially in an alpha background and transcription of alpha- and a-specific genes (MF alpha 1 and STE2) is strongly reduced. By gel retardation assays we show that the binding of the Mcm1p present in a crude protein extract from an argRIII mutant strain to the P(PAL) sequence is impaired. Sporulation of alpha/a argRIII::URA3 homozygous diploids is also affected. Overexpression of Mcm1p in an argRIII-disrupted strain restores the mating competence of the strain, the ability to form a protein complex with P(PAL) DNA in vitro, and the regulation of arginine metabolism. However, overexpression of Mcm1p does not complement the sporulation deficiency of the argRIII-disrupted strain, nor does it complement its growth defect at 37 degrees C. Western blot analysis indicates that Mcm1p is less abundant in a strain devoid of ArgRIIIp than in wild type.