Mutations Within the Minimal Replicon of Plasmid pPS10 Increase Its Host Range
Mutations in the basic replicon of the Pseudomonas plasmid pPS10 permit its establishment in E.coli, Some of the mutations affect the synthesis and/or activity of RepA, the replication protein of the plasmid, and one of the mutants, pMM141, is further analysed. Replication of pMM141 in E.coli requires functional oriV and repA sequences, and depends on the DnaA protein of the host. pMM141 can replicate in Pseudomonas.Preliminary analysis of the replication of this mutant in extracts of E.coli, shows that the plasmid replication protein RepA as well as the host proteins DnaA, DnaB, DnaG, HU, SSB, DNA gyrase and DnaK are required. In vitro replication of pMM141 can occur in the absence of transcription by host RNA polymerase. Reconstitution experiments locate the mutation in a 0.3 kb fragment of the basic replicon of pMM141 that includes the 5′ end of the repA gene and that contains the coding region for the leucine zipper motif of RepA (Giraldo et al, 1989). It is proposed that the broad host-range character of the mutant is due to the adjustment of RepA interactions to the ones required for initiation of replication in E.coli.
KeywordsReplication Protein EcoRI Fragment Replication Machinery Leucine Zipper Motif Reconstitution Experiment
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