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.     

Role of ArgR in activation of the ast operon, encoding enzymes of the arginine succinyltransferase pathway in Salmonella typhimurium

The ast operon, encoding enzymes of the arginine succinyltransferase (AST) pathway, was cloned from Salmonella typhimurium, and the nucleotide sequence for the upstream flanking region was determined. The control region contains several regulatory consensus sequences, including binding sites for NtrC, cyclic AMP receptor protein (CRP), and ArgR. The results of DNase I footprintings and gel retardation experiments confirm binding of these regulatory proteins to the identified sites. Exogenous arginine induced AST under nitrogen-limiting conditions, and this induction was abolished in an argR derivative. AST was also induced under carbon starvation conditions; this induction required functional CRP as well as functional ArgR. The combined data are consistent with the hypothesis that binding of one or more ArgR molecules to a region between the upstream binding sites for NtrC and CRP and two putative promoters plays a pivotal role in modulating expression of the ast operon in response to nitrogen or carbon limitation.     

In Saccharomyces cerevisiae, expression of arginine catabolic genes CAR1 and CAR2 in response to exogenous nitrogen availability is mediated by the Ume6 (CargRI)-Sin3 (CargRII)-Rpd3 (CargRIII) complex

The products of three genes named CARGRI, CARGRII, and CARGRIII were shown to repress the expression of CAR1 and CAR2 genes, involved in arginine catabolism. CARGRI is identical to UME6 and encodes a regulator of early meiotic genes. In this work we identify CARGRII as SIN3 and CARGRIII as RPD3. The associated gene products are components of a high-molecular-weight complex with histone deacetylase activity and are recruited by Ume6 to promoters containing a URS1 sequence. Sap30, another component of this complex, is also required to repress CAR1 expression. This histone deacetylase complex prevents the synthesis of the two arginine catabolic enzymes, arginase (CAR1) and ornithine transaminase (CAR2), as long as exogenous nitrogen is available. Upon nitrogen depletion, repression at URS1 is released and Ume6 interacts with ArgRI and ArgRII, two proteins involved in arginine-dependent activation of CAR1 and CAR2, leading to high levels of the two catabolic enzymes despite a low cytosolic arginine pool. Our data also show that the deletion of the UME6 gene impairs cell growth more strongly than the deletion of the SIN3 or RPD3 gene, especially in the Sigma1278b background.     

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.     

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.     

Gibberellin treatment stimulates nuclear factor binding to the gibberellin response complex in a barley alpha-amylase promoter.

The promoters of a majority of cereal alpha-amylase genes contain three highly conserved sequences (gibberellin response element, box I, and pyrimidine box). Recent studies have demonstrated the functional importance of four regions that either coincide with or are immediately proximal to these three conserved elements as well as an upstream Opaque-2 binding sequence. In this study, we describe the characterization of nuclear protein factors from barley aleurone layers whose binding activity toward gibberellin response complex sequences from the barley low-pl alpha-amylase gene (Amy32b) promoter is stimulated by gibberellin A3 (GA3) treatment. Barley proteins isolated from crude nuclear extracts prepared from aleurone layers incubated with or without GA3 were fractionated by anion exchange fast protein liquid chromatography and studied using band shift assays, sequence-specific competitions, and DNase I footprinting. A GA3-dependent binding activity eluting at 210 mM KCl was shown to bind specifically to the gibberellin response element and the closely associated box I. DNase I footprinting with the proteins in this fraction indicated interactions with sequences in the gibberellin response element and box I. A second DNA binding activity eluting at 310 mM KCl was present constitutively in extracts prepared from tissues incubated both in the absence and in the presence of hormone. Proteins in this fraction were able to bind to many DNA sequences and, in general, were largely nonspecific. DNase I footprinting with the proteins in this fraction indicated a large area of protection with a single unoccupied region located at the 3' end of box I. The possible function of such an activity in hormone regulation of the alpha-amylase genes is discussed.     

Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator.

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence.     

In vivo footprinting studies suggest a role for chromatin in transcription of the human 7SK gene

Mutation and deletion analyses of mammalian class III small nuclear RNA genes transcribed by RNA polymerase (pol) III have defined three functional promoter elements: a distal sequence element (DSE) at around -220, a proximal sequence element (PSE) at around -60 and a TATA box at around -30. Although binding studies have identified factors that bind to these sites in vitro, it is not known exactly how proteins interact with the promoters of these genes in vivo. In this study, we have used dimethyl sulphate and DNase I treatment of HeLa cells and nuclei, respectively, followed by linker-mediated polymerase chain reaction, to obtain in vivo footprints of proteins binding to the promoter of the Pol III-transcribed 7SK gene. Our results show that most of the characterised promoter elements of this gene are protected in vivo in these cells, and the pattern of DNase I protection suggests that a nucleosome lies between the DSE and the PSE. Methylation protection was also seen upstream of the DSE over a sequence corresponding to the binding site of a POZ domain-containing protein, ZID, which interacts with components of histone deacetylase complexes. These findings suggest that chromatin structure plays a role in the cascade of protein-DNA interactions that regulate expression of this pol III-transcribed gene.