At the replication origin of ColE1 plasmid, a persistent hybrid is formed between the primer precursor (RNA II) and its template DNA. The wild-type sequence in the region 13 to 20 bp upstream (-20 region) of the origin is required to form this persistent hybrid. While the template strand for transcription of this region, containing a stretch of six dC residues, is needed, the nontemplate strand can be deleted. Certain mutations in far upstream regions that prevent hybrid formation are suppressed by the nontemplate strand deletion. In RNA II that is forming a persistent hybrid, the region about 265 nucleotides upstream of the origin (-265 region) can also form a hybrid with the template DNA. The -265 region of RNA II that consists of a stretch of six rG residues probably interacts with the dC stretch of the -20 region in the template strand to promote hybrid formation.