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To study the functions of the mini-Pl replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer deteetable.P1 prophage is a stringently controlled plasmid replicon that is maintained at a copy number approximately equal to the number of Escherichia coli chromosomes (16). The basic plasmid replicon, the minimal region required to maintain the plasmid at its normal copy number, consists of an origin, the gene for the initiator protein RepA, and a control locus. The salient feature of the basic replicon is the presence of two sets of repeated, nearly identical 19-base-pair sequences. A set of five repeats is an essential part of the origin. A separate set of nine repeats constitutes the control locus (3, 4). The purified RepA protein binds to both sets of repeats (1). Extra copies of the repeats decrease the replication frequency, whereas deletion of the control locus increases the frequency (4, 14). Similar results were obtained for mini-F plasmids, whose genetic organization is very similar to that of mini-Pl (25). It has been proposed (25) that the origin and the control repeats compete for RepA, which has been shown to be limiting for replication (13,18), implying that the physiological concentration of the protein is so low that sequestration of a few molecules would have a significant effect on the rate of replication. In other words, the number of repeats and the number of RepA molecules are expected to be comparable in the cell. In this paper we report the measurement of RepA concentration by two approaches. These measurements are consistent with the sequestration hypothesis.The protein is also known to have two other functions. It inhibits the synthesis of the repA transcript and, when overproduced, inhibits replication (6). Knowledge of the protein concentration will thus be helpful in understanding how RepA functions. A preliminary report of some of this work has been presented (5). Protein determination. The protein concentration was determinated by using amido black as described by Schaffner and Weissman (20). All samples were brought to 1% SDS, and BSA was-used as the protein standard. The protein concentration of purified RepA as determined by amino acid analysis was about 30% less than the concentration determined by the amido black method with BSA as standard. The discrepanc...