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.     

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.     

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.     

Functional analysis of the regulatory region adjacent to the cargB gene of Saccharomyces cerevisiae. Nucleotide sequence, gene fusion experiments and cis-dominant regulatory mutation analysis

In Saccharomyces cerevisiae the expression of the cargB gene (coding for ornithine aminotransferase) is submitted to dual regulation: an induction by allophanate and a specific induction process by arginine. We have determined the nucleotide sequence of the cargB gene along with its 5' region. The coding portion of the gene encodes a protein of 423 amino acid residues with a calculated Mr value of 46049. To characterize further the regulatory mechanisms modulating the expression of the gene we have analyzed fusions of several fragments of the 5' non-coding region to lacZ, compared the 5' sequences of the cargA (coding for arginase) and cargB coregulated genes and determined the nature of two constitutive cis-dominant mutations affecting the arginine control. These approaches allowed us to define three domains in the 5' non-coding region. The upstream one is implicated in the induction by allophanate. The two other domains are involved in the specific control by arginine; the target of the ARGR gene products, that mediate a positive regulation by arginine, lies upstream of another site where a repression by the CARGRI molecule occurs. The constitutive cargB+O- mutations are located in this repressor domain. The 5' non-coding region of cargA presents the same two-domain organization. These two domains contain three sequences homologous to the cargA and cargB 5' regions.     

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.     

The nucleotide sequence of the yeast ARG4 gene

The complete nucleotide sequence of a 2296-bp DNA fragment containing the yeast (Saccharomyces cerevisiae) ARG4 gene has been determined. This gene specifies the synthesis of the arginine biosynthetic enzyme, argininosuccinate lyase (EC 4.3.2.1). The sequence contains one major open reading frame of 463 codons, giving a calculated M_r of 52010 for the protein, in good agreement with the experimentally determined value of 53 000. The sequence upstream from the ARG4 gene shares structural features in common with other yeast genes subject to general amino acid control.     

The ARG tyrosine kinase interacts with Siva-1 in the apoptotic response to oxidative stress

The Abl family of mammalian nonreceptor tyrosine kinases consists of c-Abl and ARG (Abl-related gene). Certain insights are available regarding the involvement c-Abl in the response of cells to stress. ARG, however, has no known function in cell signaling. The present studies demonstrate that ARG associates with the proapoptotic Siva-1 protein. The functional significance of the ARG-Siva-1 interaction is supported by the finding that ARG is activated by oxidative stress and that this response involves ARG-mediated phosphorylation of Siva-1 on Tyr(48). The proapoptotic effects of Siva-1 are accentuated in cells stably expressing ARG and are inhibited in ARG-deficient cells. Moreover, the proapoptotic effects of Siva-1 are abrogated by mutation of the Tyr(48) site. We also show that the apoptotic response to oxidative stress is attenuated in ARG-deficient cells and that this defect is corrected by reconstituting ARG expression. These findings support a model in which the activation of ARG by oxidative stress induces apoptosis by a Siva-1-dependent mechanism.     

Complementation of the Chlamydomonas reinhardtii arg7-8 (arg2) point mutation by recombination with a truncated nonfunctional ARG7 gene

Chlamydomonas reinhardtii arg7-8 (arg2) mutant strains carrying a hitherto undescribed mutation in their argininosuccinate lyase gene (ARG7) that leads to arginine auxotrophy have been used together with the corresponding wild-type gene as a very reliable transformation system since 1989. In this study, we finally identify the molecular nature of the arg7-8 mutation as a (6073)G to A transition in exon 9 of ARG7 leading to a (288)Gly to Ser exchange near the active site of the protein. The same mutation was found in the ARG7 genes of three commonly used C. reinhardtii laboratory strains, namely cw15-302 arg2, CC-48, and CC-1618. We did not observe exact spontaneous reversion of the arg7-8 allele in our study, but did identify two different and rare intragenic suppressor mutations, (27)Leu to Phe and (285)Tyr to Phe. In our hands, only transformation of the arg7-8 strain with a truncated nonfunctional wild-type ARG7 gene lacking 124 codons at its 5' end led to exact reversion of the mutant base (6073)A to the wild-type (6073)G, presumably by recombination. This system offers a positive selection scheme for homologous recombination (HR) and may, therefore, be useful to the methodical improvement of recombination in Chlamydomonas.     

Binding of the arginine repressor of Escherichia coli K12 to its operator sites.

In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes. In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs. The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of L-arginine. From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs. The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes. From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes. The bound repressor was shown to induce bending of argF operator DNA. The bending angle calculated from the results of gel retardation experiments is about 70 degrees and the bending center was located within the region encompassing the ARG boxes. The main features that distinguish the arginine repressor from other repressors studied in E. coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other.