Two forms of replication initiator protein: positive and negative controls.

The pir gene of plasmid R6K encodes the protein, pi, a replication and transcription factor. Two translational options for the pir gene give rise to two forms of pi protein: a 35.0-kDa form (pi35.0) and a shortened 30.5-kDa form (pi30.5). Although both proteins bind to a series of 22-bp direct repeats essential for plasmid R6K replication, only pi35.0 can bind to a site in the (A.T)-rich segment of its gamma ori and activate the gamma ori in vivo and in vitro. However, unlike pi35.0, pi30.5can inhibit in vivo and in vitro replication (activated by pi35.0). We propose that the two forms of pi might have distinct functions in replication. We show that although both forms of pi produce dimers, the nature of these dimers is not identical. The N-terminal 37 amino acid residues appear to control the formation of the more stable pi35.0 dimers, whereas another, apparently weaker interface holds together dimers of pi30.5. We speculate that the leucine zipper-like motif, absent in pi30.5, controls very specific functions of pi protein.