cis-inhibitory elements in the pT181 replication system

We report here the existence of a pair of sequence elements in plasmid cointegrates that together block the function of pT181 plasmid replication origins in cis. The study is an outgrowth of the use of plasmid pE194 as a vector for the analysis of the pT181 replication system. We have observed that whereas the isolated pT181 replication origin is fully functional when cloned to pE194, it is inactive when the entire pT181 plasmid genome is cloned. This cis-inhibition is relieved by deletion of all or part of the pE194 palA element or of the pT181 countertranscript promoter. The inhibitory effect of pE194 palA is independent of distance and orientation, whereas the inhibitory effect of the countertranscript promoter is lost when the promoter is moved to a distance of 1.5 kb from the replication origin or inverted in situ. We found that the cis-inhibited pT181 origin expresses origin-specific (Inc3B) incompatibility, which involves competition for the initiator protein. This finding suggests that the cis-inhibited origin binds the initiator protein and therefore that the inhibition affects a step in the initiation process subsequent to initiator binding.