Molecular Origins of Specificity in Protein-Nucleic Acid Interactions

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9514117 Carey The goal of this work on protein-nucleic acid interactions is to understand the molecular origins of specificity in the recognition process. How does a protein identify its target site among all available nucleic acid sites? Interest in the underlying principles of recognition motivate comparative study of different systems in order to distill out the molecular generalities that may be common among them. This work combines methods of biochemistry, biophysics, and molecular biology, with the goal of integrating structural and thermodynamic aspects of each system trying eventually to relate these aspects to the physiological role of the interaction. This proposal aims to examine molecular features responsible for specificity in two protein-nucleic acid interactions, those of bacterial arginine repressor (ArgR) and eukaryotic transcription factor IIIA (TFIIIA). These two systems share many features that recommend them as excellent examples for our studies, including the probable ability to bind both RNA and DNA and to package nucleic acids. In the case of the hexameric ArgR, important open questions concern the functional definition of the operator sequence, and the range of tolerance toward variations in both protein and DNA sequence. These issues will be approached by biochemical studies of binding to a natural operator with an extended tripartite structure, using functionally homologous but very divergent arginine repressors from E. coli and B. subtilis. Interactions of wildtype and site-directed mutant operators with each protein will be characterized by determining affinity, specificity, stoichiometry, cooperativity, and details of the DNA contacts using quantitative gel retardation and footprinting. A second aim with ArgR is to investigate a possible general regulatory role for the protein implied by some of its biochemical and physiological properties. This will be tested by using in vitro selection for ArgR-binding sequences from an E. coli genomic library. A thir d aim is to characterize a putative RNA-binding function observed in our purified samples of ArgR. To confirm that the RNA-binding moiety is ArgR, we will conduct quantitative biochemical studies of the ArgR-RNA interactions by using gel retardation and footprinting to determine affinity, specificity, stoichiometry, cooperativity, and details of the RNA contacts. In the case of TFIIIA, conflicting data in the literature indicate a need for quantitative biochemical approaches that can probe the origins of specificity in RNA binding. One hypothesis is that specificity is determined in a rapid binding equilibrium which must be studied kinetically to resolve it from subsequent particle formation. To approach this problem requires using purified recombinant components and isolated natural particles, and quantitative gel retardation and footprinting to determine the affinity, specificity, stoichiometry, cooperativity, and details of the RNA contacts in the various complexes that form during the time course of association, dissociation, competition, and exchange reaction. ***