Thr arginine repressor of E. coli, ArgR, is the transcriptional transducer of intracellular L-arginine concentration, and the master regulator of a large group of genes involved in arginine biosynthesis, the arginine regulon. ArgR is unusual among repressors because it binds DNA as a 98 kDa hexamer and functions as both a repressor and an accessory factor in resolution of plasmid ColE1 multimers by intramolecular recombination. Arginine binding increases the affinity and specificity of DNA binding by an unknown mechanism. ArgR is organized into a C-terminal domain (ArgRC) that houses the hexamerization and arginine-binding functions, and an N-terminal domain (ArgRN) which in isolation retains DNA-binding ability but is monomeric and arginine-independent. The structures of the two domains are known separately, and on the basis of these structures certain mechanisms of allosteric activation used by other regulatory proteins can be ruled out for ArgR. The separate structures of the domains, combined with biochemical and biophysical data obtained during the previous funding period on DNA binding by intact ArgR, were used to derive a model for the hexameric ArgR-DNA complex. This model and other recent progress clearly implicates subunit assembly, interdomain interactions, and conformational changes transmitted through the interdomain linker as key elements of the allosteric activation mechanism, and suggests that interactions between DNA-binding domains may confer cooperativity on the binding reaction. These hypotheses will be examined in this study with four specific aims. 1. Stoichiometry and cooperativity of DNA binding. The current model implicates only four of the six equivalent subunits of ArgR in binding to typical operator sites, which contain four half-sites arranged as tandem palindromes. The model thus implies that ArgR subunits may cooperate at pairwise or higher levels, and that a third pair of ArgR subunits is available to take up a third DNA palindrome. These features will be examined using mainly quantitative gel retardation with a set of short synthetic DNAs and cloned constructs containing various half-site arrangements to determine the stoichiometry and cooperativity of various binding modes for ArgR. The orientation of subunits on the DNA is crucial to their interactions, and will be examined following site-directed mutagenesis to permit disulfide bonding between two monomers. 2. Subunit assembly equilibria and conformational changes. Subunit assembly will be examined for the wildtype protein in the presence and absence of L-arginine by using sedimentation equilibrium analysis in the ultracentrifuge. In addition, a combination of sedimentation velocity and sedimentation equilibrium analysis will be used to probe possible global conformational changes upon L-arginine binding. 3. Role of interdomain interactions and linker in allosteric activation. Six superrepressor mutants were isolated that lie in the N-terminal domain or linker, at or near the interface with the C-terminal domain. The superrepressor phenotype suggests that transmission of the L-arg binding signal from the C-terminal domain to the DNA-binding domain is affected by the mutations. Each of these mutants will be cloned, overexpressed, and purified, and characterized by a battery of quantitative biochemical and biophysical tools to assess their effects on allosteric activation by studying their DNA- and L-arginine-binding and multimerization equilibria. 4. Global regulatory function. Functional similarities to other systems suggest that ArgR may be a previously unrecognized global regulator and/or an activator. Database searching identified several putative operator sites in genes not previously recognized as part of the arg regulon, and selective DNA binding to three of these has been confirmed. The effects of ArgR on gene expression in these three systems will be examined using b-gal fusions in various ArgR backgrounds, and the details of DNA binding will be examined in vitro.
2. Non-technical Gene expression is controlled by regulatory proteins that recognize specific DNA sequences. The mechanisms responsible for sequence-specific recognition are of fundamental interest to extend our understanding of molecular function, and also of potential practical interest for the prospect of influencing recognition events, for example in cases where expression of disease genes might be controlled. Bacterial systems provide a good model for understanding specificity because metabolically related genes are often coordinately controlled by one master regulatory protein that must recognize a group of similar but not identical DNA sequences, while maintaining the ability to reject slightly more distantly related sequences. Such systems often achieve enhanced specificity through interactions with small ligands that act as co-effectors. The molecular mechanisms through which co-effectors confer specificity are quite varied, and many apparently unique examples are known. In E. coli, the metabolism of arginine, a required amino acid, is controlled by the arginine repressor, ArgR. Arginine itself serves as a co-effector: it binds to ArgR and increases the specificity of DNA recognition by an unknown mechanism. Previous work by P.I. identified structural units within ArgR that house the various functions of the protein, and indicated that of six equivalent units, four cooperate in arginine-specific DNA recognition. This cooperation requires communication among the protein's structural units. In this study, the mechanisms of this communication will be examined. The communication will first be described in quantitative terms by studying the DNA recognition reaction directly, using methods developed previously. Interactions between units will be characterized by determining the size and shape of the molecule in solution under various conditions. A crucial interfacial region of the protein has also been identified and will be probed by studying DNA recognition using proteins that are mutated in this region. This work is expected to extend our understanding of the mechanisms leading to selective DNA binding.
