Arginine-specific repression in Saccharomyces cerevisiae: kinetic data on ARG1 and ARG3 mRNA transcription and stability support a transcriptional control mechanism.

A specific repression mechanism regulates arginine biosynthesis in Saccharomyces cerevisiae. The involvement of regulatory proteins displaying DNA-binding features and the location of an operator region between the TATA box and the transcription start of the structural gene ARG3 suggest that this mechanism operates at the level of transcription. A posttranscriptional mechanism has, however, been proposed to account for the conspicuous lack of proportionality between ARG3 mRNA steady-state levels (as determined by Northern [RNA] assays; F. Messenguy and E. Dubois, Mol. Gen. Genet. 189:148-156, 1983) and the cognate enzyme activities. In this work, we have analyzed the time course of the incorporation of radioactive precursors into ARG1 and ARG3 mRNAs and the kinetics of their decay under different regulatory statuses. The results (expressed in terms of relative mRNA levels, relative transcription rates, and mRNA half-lives) give the picture expected from a purely transcriptional control. A similar analysis of expression of the gene CPA1, for which a translational regulation by arginine has been clearly demonstrated (M. Werner, A. Feller, F. Messenguy, and A. Piérard, Cell 49:805-813, 1987), indicates that this gene is also partly regulated at the transcriptional level by the ARGR repressor system. Moreover, the half-life of CPA1 mRNA is reduced twofold in the presence of excess arginine; we suggest that this could be inherent in the mechanism of translational regulation of CPA1.

Characterization of the DNA target site for the yeast ARGR regulatory complex, a sequence able to mediate repression or induction by arginine.

We have determined the sequences and positions of the cis elements required for proper functioning of the ARG3 promoter and proper arginine-specific control. A TATA box located 100 nucleotides upstream of the transcription start was shown to be essential for ARG3 transcription. Two sequences involved in normal arginine-mediated repression lie immediately downstream of the TATA box: an essential one (arginine box 1 [AB1]) and a secondary one (arginine box 2 [AB2]). AB1 was defined by saturation mutagenesis and is an asymmetrical sequence. A stringently required CGPu motif in AB1 is conserved in all known target sites of C6 zinc cluster DNA-binding proteins, leading us to propose that AB1 is the binding site of ARGRII, another member of the C6 family. The palindromic AB2 sequence is suggested, on the basis of published data, to be the binding site of ARGRI, possibly in heterodimerization with MCM1. AB2 and AB1 correspond respectively to the 5' and 3' halves of two adjacent similar sequences of 29 bp that appear to constitute tandem operators. Indeed, mutations increasing the similarity of the other halves with AB1 and AB2 cause hyperrepression. To mediate repression, the operator must be located close to the transcription initiation region. It remains functional if the TATA box is moved downstream of it but becomes inoperative in repression when displaced to a far-upstream position where it mediates an arginine and ARGR-dependent induction of gene expression. The ability of the ARG3 operator to act either as an operator or as an upstream activator sequence, depending on its location, and the functional organization of the anabolic and catabolic arginine genes suggest a simple model for arginine regulation in which an activator complex can turn into a repressor when able to interfere sterically with the process of transcription initiation.

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.

Ets1, when fused to the GAL4 DNA binding domain, efficiently enhances galactose promotor dependent gene expression in yeast.

Ets1, the translation product of the c-ets1 proto-oncogene and the related Ets2 protein, act as sequence-specific transcriptional factors in transient transfection experiments in animal cells. We report here that in S. cerevisiae, expression of a lacZ test gene placed under the control of the GAL1 promoter is stimulated efficiently by a fusion protein in which the chicken Ets1 sequence starting from amino acid 37, is linked to the DNA binding domain of the yeast GAL4 transcriptional activator. This suggests that Ets1 contains one or more intrinsic transcription activation domain(s). However, the GAL4 integral of Ets1 fusion protein was unable to restore growth of a gal4 deletion mutant on galactose, implying that the fusion product cannot substitute for GAL4 enhancement on all GAL genes.

Two c-myb proteins differing by their aminotermini exhibit different transcriptional transactivation activities (yeast/reporter-effector system).

We assayed in the yeast S. cerevisiae the transcriptional transactivation activity of the c-myb products encoded by a normal thymus cDNA and of an aminoterminally truncated version of it (minus 58 amino acids) corresponding to the cDNAs isolated from lymphoma and leukemia cells from different origins. Both proto-oncogene products were expressed under the control of the galactose inducible GAL10 promoter. The reporter system used to monitor the transactivation potential of the myb products consisted of a CYCl-lacZ gene fusion in which the UASCYC signals were replaced by one or multiple copies of the myb recognition element (mRE). As shown by Northern blot analyses and by primer extension experiments both c-myb products increase the level of beta-galactosidase transcription. Interestingly, the c-myb product corresponding to lymphoma cDNAs stimulates transcription four to five times more efficiently than does the normal thymic c-myb product.

The carboxy-terminal domain of c-Myb activates reporter gene expression in yeast.

We have shown previously that c-myb expressed in the yeast S. cerevisiae mediated efficient transcriptional activation of reporter genes designed with specific Myb Recognition Elements (MRE's), confirming that this proto-oncogene is able to function as a regulator of transcription in that heterologous context. Here we show that in yeast, as in higher eucaryotic cells, the central domain of c-Myb displays transactivating capacity. In yeast, however, the carboxy-terminal region, defined as a negative regulatory domain in higher cells, activates transcription as well and appears to be a more potent transactivating domain than the central domain itself. Within this region two domains, namely C1 and C2, have been defined that contribute about equally to the activity of the carboxy-terminal region. C1 spans the sequences missing in AMV v-myb while C2, which contains the leucine-zipper motif is specifically absent in the E26 v-myb in addition to C1. The c-Myb DNA-binding domain itself has no effect on the level of transcription in yeast. We also show that AMV v-Myb stimulates gene expression in yeast with about half the efficiency of full length c-Myb. The fact that the carboxy-terminal region either stimulates or inhibits transactivation properties of c-Myb, depending on the cellular context, stresses the participation of putative c-Myb partner proteins in Myb regulated processes and reopens the question of whether the oncogenic activation of c-myb is indeed due to the increased transactivation capacity of its onco derivatives.

Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli. I. Primary structure of a pyrI gene encoding a modified regulatory subunit.

In a previous article, we have identified a lambda bacteriophage directing the synthesis of a modified aspartate carbamoyltransferase lacking substrate-co-operative interactions and insensitive to the feedback inhibitor CTP. These abnormal properties were ascribed to a mutation in the gene pyrI encoding the regulatory polypeptide chain of the enzyme. We now report the sequence of the mutated pyrI and show that, during the generation of this pyrBI-bearing phage, six codons from lambda DNA have been substituted for the eight terminal codons of the wild-type gene. A model is presented for the formation of this modified pyrI gene during the integrative recombination of the parental lambda phage with the Escherichia coli chromosome. An accompanying paper emphasizes the importance of the carboxy-terminal end of the regulatory chain for the homotropic and heterotropic interactions of aspartate carbamoyltransferase.

Escherichia coli and Saccharomyces cerevisiae acetylornithine aminotransferase: evolutionary relationship with ornithine aminotransferase.

Genes argD and ARG8, encoding the acetylornithine aminotransferase (ACOAT) subunit in Escherichia coli and Saccharomyces cerevisiae, respectively, have been cloned and sequenced. The deduced amino acid sequences show substantial similarity. Moreover, they resemble ornithine aminotransferase (OAT) sequences (i.e., those from yeast, rat and man); the observed similarities are statistically significant, indicating that the enzymes are homologous. However, in contrast to OATs, which appear to be substrate (i.e., ornithine)-specific, S. cerevisiae ACOAT transaminates ornithine about as efficiently as E. coli does. The evolutionary relationship between ACOATs and OATs is discussed in terms of substrate ambiguity.

Cloning and endonuclease restriction analysis of argF and of the control region of the argECBH bipolar operon in Escherichia coli.

A 1.8 kb DNA fragment, liberated by endonuclease HindIII, contains the control region of the argECBH bipolar operon near one end and the weak secondary promoter of argH at the other extremity; it has been cloned in plasmid pBR322. The same plasmid vector has been used to clone the argF gene liberated from the chromosome by endonuclease BamHI. Restriction patterns for the two hybrid plasmids have been determined, using enzymes AluI, BglI, EcoRI, HaeIII, HincII, HindIII, HpaI and II, PstI and SalI. Two AluI sites situated on either side of and close to a HincII target delineate two short fragments covering the whole of the argECBH control region. The argF control elements are located in a region accessible to further dissection by BamHI, EcoRI, PstI and HindIII. Carriers of the argF plasmid produce extremely high amounts of ornithine carbamoyltransferase, a feature useful for purification of this enzyme.

Sequences of the genes encoding argininosuccinate synthetase in Escherichia coli and Saccharomyces cerevisiae: comparison with methanogenic archaebacteria and mammals.

The nucleotide (nt) sequences of the genes encoding argininosuccinate synthetase from Escherichia coli K-12 (argG) and Saccharomyces cerevisiae (ARG1) were determined. The deduced amino-acid sequences were compared to each other and to their counterparts in two methanogens and in mammals. Three regions are highly conserved. Two of them appear to contain possible Walker-type nt-binding sites [Walker et al., EMBO J. 1 (1982) 945-951] and are therefore candidates for ATP-binding sites. The third region shows some similarity to a short portion of the N-proximal part of the PurA enzyme which catalyses an analogous reaction.

Studies on the bipolar argECBH operon of E. coli: characterization of restriction endonuclease fragments obtained from gammadargECBH transducing phages and a ColE1 argECBH plasmid.

The isolation of a new type of gamma transducing phage carrying the bipolar argECBH operon of E. coli K12 is described. The argECBH segment is inserted in the phage in a direction which is opposite from that of previously isolated argECBH-carrying phages. A colE1 argECBH plasmid has been constructed. DNA fragments resulting from digestion of these genetic elements with Eco RI and Hind III restriction enzymes have been characterized by agarose gel electrophoresis and electron microscopy, including hetero-duplex analysis. Two fragments are of special significance for studies on the control of arginine synthesis, one of length 9.8 kilobases carrying the whole argECBH region, the other of length 2 kilobases carrying most or all of the control region between argE and argC.

Nucleotide sequence of the ARG3 gene of the yeast Saccharomyces cerevisiae encoding ornithine carbamoyltransferase. Comparison with other carbamoyltransferases.

The complete nucleotide sequence of the ARG3 structural gene encoding the monomer of the trimeric ornithine carbamoyltransferase (OTCase) (EC 2.1.3.3) has been determined. It consists of 338 codons with a corresponding molecular mass of 37842 Da. Comparing OTCases from Escherichia coli, yeast, Aspergillus, rat and man emphasizes peculiarities of the yeast enzyme but also brings to light an important degree of conservation between these proteins. Comparing the various OTCases with E. coli aspartate carbamoyltransferase (ATCase) (EC 2.1.3.2) confirms the evolutionary relationship previously noted between the two types of carbamoyltransferases and points to residues probably involved in catalysis and structural folding in OTCases.

Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease.

Kinetic and genetic evidences are presented to show that, in addition to specific amino acid permeases, Saccharomyces cerevisiae has a general amino acid permease which catalyzes the transport of basic and neutral amino acids, but most probably not that of proline. The general amino acid permease appears to be constitutive, and its activity is inhibited when ammonium ions are added to the culture medium. A mutant which has lost the general amino acid permease activity was isolated. Its mutation, named gap (general amino acid permease), is not allelic to the aap (amino acid permease) mutation of Surdin et al., which has a quite different phenotype and cannot be considered as having selectively lost the general amino acid permease activity.

Tandem and inverted repeats of arginine genes in Escherichia coli: structural and evolutionary considerations.

Duplications of arg genes produced in the Rec+ and in the recA genetic backgrounds are shown by heteroduplex analysis to be strictly tandem at the level of resolution of this technique. The formation of these particular rearrangements therefore does not require the inclusion of transposons or other sequences of an appreciable size in their final structure. Duplications of short segments (about 2,000 nucleotides) appear unexpectedly stable when compared with duplications of longer segments (about 10,000 nucleotides). One of the structures analyzed displays two inversely repeated argE genes rearranged into an artificial divergent operon. The bearing of this observation on the origin of bipolar operons, of "mirror-image" map symmetries and on the production of inverted repeats in general, is discussed.

Arginine repression of the Saccharomyces cerevisiae ARG1 gene. Comparison of the ARG1 and ARG3 control regions.

The Saccharomyces cerevisiae ARG1 gene coding for argininosuccinate synthetase has been isolated and the nucleotide sequence of both its control region and of its amino terminal end coding region determined. The startpoint of transcription was established by S1-mapping and reverse transcriptase procedures. Northern blot hybridizations showed that whereas arginine-specific repression reduced the enzyme activity fivefold, it did not reduce the steady state level of the corresponding messenger in proportion; by analogy with the coregulated ARG3 gene, this result suggests a post-transcriptional regulatory mechanism. In contrast, proportionally between enzyme activity and mRNA content was observed under conditions where general amino acid control (known to be transcriptional) was operating. Comparing the 5' untranscribed domains of ARG1 and ARG3 revealed a first region of homology between the TATA box and the transcription startpoint. In this region a 10 bp (ARG3) or 11 bp (ARG1) central box is flanked by two segments which, by mutation, have been shown to be part of the ARG operator (Crabeel et al. 1985). The repressor is assumed to bind at this primary target site prior to establishing contacts with the proximal part of the nascent mRNA molecule (Crabeel et al. 1985). By in vitro directed deletion mutagenesis we show that the central conserved box of ARG3 is not essential for arginine-specific repression to occur. Another region of homology was found in the leader part of the messenger RNA; deletion of this region causes a small reduction in ARG3 expression but also does not alter regulation. Neither of these two regions are thus part of the primary repressor target site. In addition, in terms of post-transcriptional regulation, the latter result indicates that no sequence specificity is required in the RNA recognition step.

Cloning arg3, the gene for ornithine carbamoyltransferase from Saccharomyces cerevisiae: expression in Escherichia coli requires secondary mutations; production of plasmid beta-lactamase in yeast.

The yeast arg3 gene, coding for ornithine carbamoyltransferase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3), has been cloned on a hybrid pBR322-2-micrometers plasmid. The cloned gene gives a normal regulatory response in yeast. It is not expressed at 35 degrees C when a mutation preventing mRNA export from the nucleus at this temperature is included in the genetic make-up of the carrier strain. In Escherichia coli, no functional expression can be observed from the native yeast arg3 gene. The study of a mutant plasmid (M1) producing low levels of yeast carbamoyltransferase in E. coli has permitted the localization and orientation of arg3 on the plasmid. The mutation involved is a deletion that alters the regulatory response of arg3 in yeast. The plasmid bla gene produces detectable amounts of beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) in yeast: the data provide an estimate of the beta-lactamase activity associated with one exemplar of the plasmid expressing arg3 (0.6 units).

The promoter region of the arg3 gene in Saccharomyces cerevisiae: nucleotide sequence and regulation in an arg3-lacZ gene fusion.

We have determined the DNA sequence for the 5' end of the arg3 gene of Saccharomyces cerevisiae, including part of the coding region and the 200 nucleotides immediately upstream. A promoter-deletion mutant was found to have lost all of the sequence lying normally in front of the gene except for the 33 nucleotides preceding the AUG codon. The role of the 5' domain in initiation and regulation of arg3 transcription was assessed by a gene fusion experiment. The Escherichia coli lacZ gene, was truncated of the eight amino-terminal codons substituted in vitro, on a 2mu plasmid, for the carboxy-terminal and 3'-flanking regions of arg3, leaving only the first 19 proximal codons and approximately 1600 nucleotides of the region preceding arg3 on the yeast chromosome. The fused gene was expressed in phase and was still submitted to the two mechanisms regulating the wild-type arg3 gene: the general, probably transcriptional control of amino acid biosynthesis and the specific, apparently post-transcriptional control mediated by the products of the argR genes. These results suggest a determining role for the 5' end portion of the arg3 messenger in the specific arginine-mediated control mechanism.

Control-mechanisms acting at the transcriptional and post-transcriptional levels are involved in the synthesis of the arginine pathway carbamoylphosphate synthase of yeast.

In Saccharomyces cerevisiae, the synthesis of the arginine pathway enzyme carbamoylphosphate synthase (CPSase A) is subject to two control mechanisms. One mechanism, the general control of amino acid biosynthesis, influences the expression of both CPA1 and CPA2 genes, the structural genes for the two subunits of the enzyme. The second mechanism, the specific control of arginine biosynthesis, only affects the expression of CPA1. To study these mechanisms in more detail, we have cloned the CPA1 and CPA2 genes and used their DNA to measure the CPA1 and CPA2 mRNA content of cells grown under various conditions. A close coordination was observed in the variation of the levels of CPA1 and CPA2 mRNAs and polypeptide products under conditions where the general control of amino acid biosynthesis operates. In contrast, little correlation was found between the levels of CPA1 mRNA and the corresponding protein for conditions affecting repression by arginine: the total amplitude of variation was 6-fold higher for the CPA1 protein than for the CPA1 messenger transcript. Such findings are consistent with the conclusion that the general control operates at the transcriptional level and that the specific arginine control acts primarily at a post-transcriptional level.

A new yeast metabolon involving at least the two first enzymes of arginine biosynthesis: acetylglutamate synthase activity requires complex formation with acetylglutamate kinase.

Open reading frame YJL071W of Saccharomyces cerevisiae was shown to be ARG2 and identified as the structural gene for acetylglutamate synthase, first step in arginine biosynthesis. The three Ascomycete acetylglutamate synthases characterized to date appear homologous, but unlike the other enzymes of the yeast arginine biosynthesis pathway, they showed no significant similarity to their prokaryotic equivalents. The measured synthase activity did not increase with the number of ARG2 gene copies unless the number of ARG5,6 gene copies was increased similarly. ARG5,6 encodes a precursor that is maturated in the mitochondria into acetylglutamate kinase and acetylglutamyl-phosphate reductase, catalyzing the second and third steps in the pathway. The results imply that the synthase must interact stoichiometrically in vivo with the kinase, the reductase, or both to be active. Results obtained with synthetic ARG5 and ARG6 genes suggested that both the kinase and the reductase could be needed. This situation, which has completely escaped notice in yeast until now, is reminiscent of the observation in Neurospora crassa that nonsense arg-6 kinase/reductase mutants lack synthase activity (Hinde, R. W., Jacobson, J. A., Weiss, R. L., and Davis, R. H. (1986) J. Biol. Chem. 261, 5848-5852). In immunoprecipitation experiments, hemagglutinin-tagged synthase coprecipitated with a protein proven by microsequencing to be the kinase. Western blot analyses showed that the synthase has reduced stability in the absence of the kinase/reductase. Our data demonstrate the existence of a new yeast arginine metabolon involving at least the first two, and possibly the first three, enzymes of the pathway. Hypotheses regarding the biological significance of this interaction are discussed.

A strong carbon source effect is mediated by pUC plasmid sequences in a series of classical yeast vectors designed for promoter characterization.

While using YIp356 and YEp356R lacZ reporter plasmids, we found lacZ expression driven by the ARG2 promoter to be much higher in cells grown on a non-glucose carbon source than in glucose-grown cells (5-10-fold higher on galactose and up to 40-fold higher on ethanol). Furthermore, expression increased 30-fold upon shifting from a high-glucose to a low-glucose medium. This carbon source regulation requires Snf1p and possibly Ssn6p. It appears, however, to be artefactually mediated by plasmid sequences located upstream from the multicloning site. This emerged from the following observations: (a) the derepressive effect disappears if any extra piece of DNA is inserted upstream from the ARG2 promoter; and (b) similar derepression on low glucose is observed with another yeast promoter (ARG11), provided that the flanking 5' region is short. We determined that the cis-elements responsible for this physiologically irrelevant glucose regulation are located between positions 636 and 879 of the pUC18 DNA sequence.