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

Abstract

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.