Initiation of ColE1 DNA replication can be demonstrated with a mixture of E. coli RNA polymerase, ribonuclease H (RNase H) and DNA polymerase I. Transcription of ColE1 DNA starts at two sites in a region required for maintenance of the plasmid. Certain transcripts that start at one of the sites (primer promoter) can be cleaved by RNase H and then act as primer for DNA replication. Transcription from the other site produces an RNA-108 nucleotides long (species I or RNAI). If purified RNA I is added to the transcription reaction containing RNase H, formation of primer is inihibited. The inhibition of primer formation by RNA I is incompatibility specific. The step that is sensitive to inihibition is probably formation of the hybrid between the primer precursor and the template.
Mutans of pNT7, a small derivative of ColE1, that have altered incompatibility properties have been isolated. Each mutant has a single base change at or near the center of at least one of the three palindromes in the region that specifies both the primer RNA and RNA I. The single base changes affect both the ability of RNA I to inhibit primer formation and, as well, the sensitivity of primer formation to inhibition by RNA I. RNA I hybridizes to transcripts originating at the primer promoter and the rate of hybridization is reduced by the single base changes. The copy number of a plasmid is determined by the extent of inhibition of primer formation by its own RNA I and incompatibility between two plasmids can be attributed to inhibition of primer formation of at least one of the plasmids by the RNA I of the other.