Plasmid replication functions. VII. Electron microscopic localization of RNA polymerase binding sites in the replication control region of plasmid R6-5.

RNA polymerase binding sites on the R6-5 miniplasmid derivative, plasmid pKT401, were mapped by electron microscopy of DNA:RNA polymerase complexes formed with both circular-supercoiled and restriction endonuclease-linearized plasmid DNA molecules. Of eight specific binding sites on pKT401 that were identified, three were found to be in the P-6 fragment of the plasmid replication region, three in the Tn3 element, and two in other parts of the plasmid molecule. Binding sites 1 and 3 in the P-6 fragment are most probably the promoters of the copB and copA/incA plasmid replication control genes, respectively, whereas site 2 in this fragment appears to be the promoter of the essential replication gene, repA. The location of these promoters in relation to the site of action of the plasmid replication control elements, copT, and the origin of replication, oriV, suggests that replication control may be effected by regulation of transcription events initiated at site 2, or of the activity of transcripts initiated from this site, i.e., by regulation of the expression of the repA gene or another function dependent upon these events.