Primary structure of the essential replicon of the plasmid pSC101.

Abstract

The replicon of the low copy number plasmid pSC101 has an obligatory requirement for the dnaA initiator protein of Escherichia coli and a plasmid-encoded initiator protein. The cistron of the plasmid-encoded initiator was identified by DNA sequence analysis. Fusion of the initiator cistron with the lacZ gene of E. coli yielded a fusion protein of .apprxeq. 150 kilodaltons, confirming that the open reading frame detected by DNA sequence analysis actually encoded a 37.5-kilodalton protein. Deletion of 26 amino acid residues from the COOH terminus of the plasmid initiator abolished autonomous replication from pSC101 origin. By in vitro deletion analysis, it was shown that, although sequences downstream from the initiator cistron are dispensable, a maximum of 400 base pairs immediately upstream from the NH2-terminal region of the initiator is necessary for plasmid replication. These upstream sequences contain an A + T-rich region and 3 tandem repeats of a 21-base pair sequence; these features are characteristics of other replication origins.