THE DIMERIZATION DOMAIN OF R6K PLASMID REPLICATION INITIATOR PROTEIN-PI REVEALED BY ANALYSIS OF A TRUNCATED PROTEIN

Replication of plasmid R6K is controlled by the homodimeric initiator protein pi, which binds to seven 22-bp direct repeats (iterons) in the gamma-origin. One of the genetically engineered pi variants (Delta C1 64 pi) contains only the 164 N-terminal amino acids (aa) of the 305-aa pi molecule. This truncated pi polypeptide retains the ability to fun ction as a specific inhibitor of R6K replication in vivo, though it ne ither drives replication, nor binds to iterons [Greener et al., Mol. G en. Genet. 224 (1990) 24-32]. In order to define the region of pi, res ponsible for dimerization, we have performed chemical crosslinking exp eriments with purified Delta C164 pi and shown that this polypeptide i s dimeric. We did not observe an exchange between protein monomers upo n mixing of wild-type pi and Delta C164 pi homodimers. However, hetero dimers, as well as each type of homodimers, were formed when these pol ypeptides refolded after guanidine hydrochloride treatment. Thus, both dimerization and dimer stability are determined by the N-terminal dom ain of pi. We speculate that these properties might depend on the leuc ine zipper and RGD motifs that have been identified in the two regions of the N-terminal domain of pi.