FUNCTIONAL FEATURES OF AN SSI SIGNAL OF PLASMID PGKV21 IN ESCHERICHIA-COLI

A single-strand initiation (ssi) signal,vas detected on the Lactococcu s lactis plasmid pGKV21 containing the replicon of pWV01 by its abilit y to complement the poor growth of an M13 phage derivative (M13 Delta lac182) lacking the complementary-strand origin in Escherichia coli. T his ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragm ent and located within the 109 nt upstream of the nick site of the put ative plus origin. SSI activity is orientation specific with respect t o the direction of replication. We constructed an ssi signal-deleted p lasmid and then examined the effects of the ssi signal on the conversi on of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. c oli, this ssi signal directs its lagging-strand synthesis as a minus o rigin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that B . coli RNA polymerase directly recognizes the 229-nt ssi signal and sy nthesizes primer RNA dependent on the presence of E. coli single-stran ded DNA binding (SSB) protein. This region contains two stem-loop stru ctures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E, coli RNA polymerase, suggest ing that stem-loop I portion is essential for priming by E. coli RNA p olymerase on the SSB-coated single-stranded DNA template.