title: Effects of point mutations on formation and structure of the RNA primer for ColE1 DNA replication.
abstract: 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.
mesh_terms: D003086:Bacteriocin Plasmids; D001483:Base Sequence; D004261:DNA Replication; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D009154:Mutation; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics*; D016914:Ribonuclease H; D013698:Templates, Genetic; D014158:Transcription, Genetic
chemical_list: D012329:RNA, Bacterial; D004722:Endoribonucleases; D016914:Ribonuclease H