Effects of point mutations on formation and structure of the RNA primer for ColE1 DNA replication.

We report mutations affecting several steps of CoIE1 primer formation. Primer precursor forms the hybrid with the template DNA and is cleaved by RNAase H to form the primer. Point mutations at positions -264, -265, -268, and -308 (base pairs upstream of the replication origin) reduce efficiency of hybrid formation. A suppressor mutation at -18 increases the efficiency for a mutant transcript and reduces that for a wild-type transcript. Therefore, hybrid formation probably starts after transcription passes this position. A mutation at -10 affects the site of cleavage by RNAase H. The site appears to be determined by the distance from a stem-loop structure immediately upstream. A double mutation at -186 and -188 results in formation of an RNA secondary structure that prevents use of an RNAase H-cleaved transcript as primer.