ALTERED (COPY-UP) FORMS OF INITIATOR PROTEIN-PI SUPPRESS THE POINT MUTATIONS INACTIVATING THE GAMMA-ORIGIN OF PLASMID R6K

The R6K gamma origin core contains the P2 promoter, whose -10 and -35 hexamers overlap two Of the seven binding sites for the R6K-encoded pi protein, Two mutations, P2-201 and P2-203, which lie within the -35 r egion of P2, are shown to confer a promoter-down phenotype. We demonst rate here that these mutations prevent replication of a gamma origin c are plasmid, To determine whether or ndt the reduced promoter activity caused by these mutations is responsible for their effect on replicat ion, we generated two new mutations (P2-245-6-7 and P2-246) in the -10 hexamer of the P2 promoter. Although these new mutations inhibit P2 a ctivity as much as the P2-201 and P2-203 mutations, they do not preven t replication of the gamma origin core. Therefore, activity of the P2 promoter does not appear to be required for replication. We also show that the inability of the gamma origin to function in the presence of the P2-201 and P2-203 mutations is reversed by the hyperactive variant s of pi protein called copy-up pi. This suppression occurs despite the fact that in vivo dimethyl sulfate methylation protection patterns of the gamma origin iterons are identical in cells producing wild-type p i and those producing copy-up pi variants. We discuss how the P2-201 a nd P2-203 mutations could inhibit replication of the gamma origin core and what mechanisms might allow the copy-up pi mutants to suppress th is deficiency.