The long-term aim of the experiments proposed here is a comprehensive description of the molecular rules governing the frequency of initiation of DNA replication in a stably inherited replicon. Our model system for these experiments is the E. coli plasmid ColE1 and its derivatives. Experiments are described which analyze the three plasmid-encoded elements known to play central roles in replication control of ColE1: the RNA primer for replication; a small RNA, designated RNA1, that negatively modulates the frequency of processing of the primer by a unique RNA-RNA interaction; and the rop gene product, a small polypeptide that acts as a second negative control element on primer formation. A central tool for these experiments is the isolation of mutants in these control elements that affect plasmid copy number and/or incompatibility properties. Procedures are described to isolate mutants in each of the control elements and to produce these altered elements in large quantities. The interaction between RNA1 and primer precursor is extremely sensitive to perturbations in the RNA secondary and tertiary structures of these molecules. A major aim of this proposal is to define the structural domains of wild-type primer RNA and compare these domains to those displayed by specific primer mutants. Methods are described for the isolation and characterization of RNA1 mutants and for analysis of RNA1-primer interaction in vitro. Experiments are proposed to investigate the mechanisms by which the rop polypeptide affects primer formation. Methods for isolation of rop structural mutations and mutations in the rop-interaction site are proposed. Efforts will be made to isolate a rop-RNA1 ribonucleoprotein complex which may play a role in primer interaction. The physical interaction between mutant rop and mutant RNA1 species will also be explored, as will the structural consequnces for primer RNA when produced in the presence of rop-RNA1. Finally the roles of specific host genes in modulating the synthesis or activity of the ColE1 control elements will be explored. We will pursue the possibility that the E. coli dam methylase is involved in rop function and that the dnaA gene is a negative control element for synthesis of the RNA1 gene.
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