Our long term goals are to understand the mechanism of control of initiation of DNA replication in the plasmids R6K and RK2 and to further investigate the alternative pathways of replication initiation in RK2 recently discovered by us. Our specific goals are as follows: (a) to investigate DNA looping in vivo and in vitro in the activation of beta and alpha origins of R6K by initiator protein bound to the gamma-enhancer; (b) to further investigate the interaction of silencer RNA with activator RNA and ori DNA and the role of initiator protein in these interactions; (c) to use and antisence strategy to identify and map the activator (primer) RNAs of alpha, beta and gamma origins. Using various labeling techniques, maps the RNA-DNA junctions in these putative primer RNAs; (d) to use purified host proteins and in vitro replication system to reconstitute R6K replication initiation in vitro; (e) to purify the trfA, K or B and 13 Kd proteins of RK2 to homogeneity and to study their interactions with the ori; (f) to elucidate biochemcial properties of these proteins by using in vitro replication system. The analysis of the dnaA dependent and independent pathways of initiation in RK2, recently discovered by us, should illuminate the various strategies used by the broad host range plasmid to initiate and control its replication in gram negative hosts.