GENETIC ANALYSIS OF A TRANSLATIONAL REPRESSOR

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Protein-RNA interactions are increasingly recognized as important to a variety of essential cellular activities. The coat protein of the RNA bacteriophage MS2 is a translational repressor. At late times of infections coat protein binds a specific stem-loop structure in the viral mRNA and responses synthesis of replicase. Although we already know many of the important functional components of the 21 nucleotide operator sequence, similar information about coat protein is unavailable. We have constructed a genetic system in which coat protein represses synthesis of a replicase-beta-galactosidase fusion protein. This permits us to conveniently apply the methods of molecular genetics to determine the molecular basis of specific protein-RNA interactions using coat protein as a model system. Specifically we want to accomplish the following objectives: 1. Define amino acid residues necessary for coat protein's RNA binding function by isolating and characterizing a number of mutations that alter the strength and/or specificity of operator binding. We will isolate mutations of the following type: a. repressor-defective mutations, b. intragenic suppressors of repressor-defective mutations, and c. coat protein suppressors of operator-constitutive mutations. 2. The RNA binding properties of mutant coat proteins will be characterized: a. in vivo by measuring repression of synthesis of the hybrid replicase-beta-galactosidase enzyme, b. in vitro determination of RNA binding affinity, and c. by chemical modification studies which will determine nucleotides in the operator that are important for recognition by wild-type and mutant coat proteins. 3. Test genetically and biochemically the idea that the formation of a transient covalent bond between a coat protein sulfhydryl and an essential pyrimidine in the operator is a component of the coat protein-RNA interaction.