CHAPERONE-MEDIATED REDUCTION OF REPA DIMERIZATION IS ASSOCIATED WITH REPA CONFORMATIONAL CHANGE

RepA, the initiator protein of plasmid P1, binds to multiple sites (it erons) in the origin, The binding normally requires participation of c haperones, DnaJ, DnaK and GrpE. When purified, RepA appears dimeric an d is inactive in iteron binding, On reaction with chaperones, a specie s active in iteron binding is formed and found to be monomeric. To tes t whether the chaperones can reduce dimerization, RepA was used to rep lace the dimerization domain of the lambda repressor. The hybrid prote in repressed the lambda operator efficiently, indicating that RepA can dimerize in vivo. A further increase in repressor activity was seen i n dnaJ mutant cells, These results are consistent with a chaperone-med iated reduction of RepA dimerization, We also found that RepA mutants defective in dimerization still depend on DnaJ for iteron binding, Con versely, RepA mutants that no longer require chaperones for iteron bin ding remain dimerization proficient, These results indicate that the c haperone dependence of RepA activity is not solely owing to RepA dimer ization, Our results are most simply explained by a chaperone-mediated conformational change in RepA protomer that activates iteron binding. This conformational change also results in reduced RepA dimerization.