THE CENTRAL REGION OF REPE INITIATOR PROTEIN OF MINI-F PLASMID PLAYS A CRUCIAL ROLE IN DIMERIZATION REQUIRED FOR NEGATIVE REPLICATION CONTROL

The RepE protein (251 residues, 29 kDa) of mini-F plasmid, mostly foun d as dimers, plays a key role in mini-l; replication. Whereas monomers bind to the origin to initiate replication, dimers bind to the repE o perator to repress its own transcription. Among the host factors requi red for mini-F replication, a;set of molecular chaperones (DnaK, DnaJ and GrpE) is thought to facilitate monomerization of RepE dimers. To f urther understand the structural basis of functional differentiation b etween the two forms of RepE, we examined the region(s) critical for d imerization by isolation and characterization of RepE mutants that wer e defective in autogenous repressor function. Such mutations were isol ated from two separate regions of RepE, the central region (residues 1 11 to 161) and the C-terminal region (residues 195 to 208). The centra l region overlapped the region where the chaperone-independent copy-up mutations were previously isolated (residues 93 to 135). Likewise the mini-F mutant plasmids, carrying the mutations in the central region, could replicate in a dnaK null mutant host. One of them, S111P (111th serine changed to proline), showed a very high origin-binding activit y vis-a-vis a severely reduced operator-binding activity, much Like th e RepE54.(R118P) mutant previously shown to form only monomers. Gel fi ltration and chemical crosslinking studies with purified RepE revealed that S111P primarily formed monomers, whereas other mutant proteins f ormed mostly dimers. On the other hand, analysis of deletion mutants r evealed that the N-terminal 42 and the C-terminal 57 residues were dis pensable for dimerization. Thus, the region spanning residues 93 to 16 1 of RepE (including Ser111 and Arg118) appeared to be primarily invol ved in dimerization, contributing to the negative regulation of plasmi d replication. (C) 1997 Academic Press Limited.