The iteron bases and spacers of the P1 replication origin contain information that specifies the formation of a complex structure involved in initiation.

The origin of replication of the P1 plasmid contains five direct, imperfect repeats (iterons) of a 19 bp sequence that binds the P1-encoded RepA initiator protein. RepA binding to these iterons triggers origin initiation and represses transcription from the repA promoter that is nested within the iterons. The origin iterons were replaced with ligated oligonucleotides that insert five perfect 19 bp repeats with identical spacer sequences. This eliminates the natural variation in the iteron and spacer sequences and removes the repA promoter. The reconstructed origin is functional, showing that the repA promoter is not essential for origin function. The method used to make the reconstructed origin allows substitution of identical iterons with altered sequence or spacer length. Single changes of conserved iteron bases gave reduced or non-existent origin activity, as did an increase in spacer length. Like the wild type, most of these mutant arrays retain avid primary binding activity for the RepA protein. However, although the wild-type arrays readily form a mature complex in which all iterons are saturated, the most replication-defective mutants were completely unable to do this, even at very high RepA concentrations. It appears that iteron spacing and contacts involving at least three of the conserved iteron bases play an important role in the assembly of the mature structure in which all sites are occupied. A model is presented in which an allosteric interaction between the DNA site and protein is needed for the saturated, mature complex required for initiation.

Control of P1 plasmid replication by iterons.

The incA locus of plasmid P1 controls plasmid copy number by inhibiting the replication origin, oriR. Both loci contain repeat sequences (iterons) that bind the P1 RepA protein. Regulation appears to occur by contact of incA and oriR loci of daughter plasmids mediated by RepA-bound iterons. Synthetic incA iteron arrays were constructed with altered numbers, sequences or spacing of iterons. Using these in in vitro and in vivo assays, we examined two models: (i) that the origin and incA loci form a stable 1:1 complex in which multiple iterons of each locus are paired with those of the other, and (ii) that individual incA iterons act as freely diffusing nucleoprotein units that contact origin iterons in a random and dynamic fashion. The data presented here strongly favour the latter case. The origin, with its five iterons, acts as a target but not as an effector of regulation. We present a model for replication control based on random, dynamic contacts between incA iterons and the origin. This system would display randomness with respect to choice of templates and timing of initiation if multiple replicon copies were present, but would tend to act in a machine-like fashion in concert with the cell cycle if just two copies were present in a dividing cell.

Unique sequence requirements for the P1 plasmid replication origin.

We have carried out a detailed genetic analysis of the P1 plasmid replication origin and shown that it has four major structural requirements: the DnaA box, a series of five 7-base pair (bp) repeats, a GC-rich spacer and five 19-bp repeats that bind the P1 RepA protein. The origin requires the DnaA protein and its recognition sequence (the DnaA box). However, although five boxes are present in two separate blocks in the wild type, just one, placed either to the left or right of the core origin sequences, is sufficient for function as long as the box conforms exactly to the proposed consensus. Each of the five 7-bp repeats that constitute the core of the origin is required; mutations within any of the first six base pairs can block origin function. The required bases include, but are not limited to, those constituting dam methylation sites. Also essential is a 39-bp GC-rich sequence. We show this to be a spacer of critical length that separates the 7-bp repeats from the last required region; a series of 19-bp repeats that bind the P1 RepA initiator protein.

A single DnaA box is sufficient for initiation from the P1 plasmid origin.

The P1 plasmid replication origin requires the host DnaA protein for function. Two DnaA-binding boxes lie in tandem within the previously defined minimal origin, constituting its left boundary. Three more boxes lie 200 base pairs to the right of these, in the leader region for the P1 repA gene. We show that either set alone is active for origin function. One of the two origin boxes is relatively inactive. Constructs with just one of the five boxes are active for specific origin function as long as the box conforms exactly to the published consensus. This single consensus box is functional when placed either to the left or right of the core origin sequences. The flexibility shown by this system suggests that the boxes play a role different from those in the host oriC origin, where the number and position of boxes are critical.

Protein-DNA interactions in regulation of P1 plasmid replication.

The P1 RepA protein appears to play three roles in P1 plasmid replication: acting at the origin both as a specific initiator and as a repressor of transcription, and interacting with the copy-control locus incA to bring about a negative control of initiation. We have used the DNase I footprinting technique to show that RepA binds specifically to repeat units of a 19-base-pair consensus sequence present in both the origin and incA control regions. RNA polymerase was shown to bind to two specific regions within the origin repeats. One of these constitutes the known promoter sequence for the repA gene. We show evidence that the polymerase can be efficiently displaced from the promoter by subsequent RepA binding, thus providing a direct mechanism for RepA autoregulation. Under the conditions used, there were no obvious differences in the affinities of individual repeat sequences for the purified protein.