A Single Base-pair Alteration is Responsible for the DNA Overproduction Phenotype of a Plasmid Copy-number Mutant

The Cop- plasmid pOP1 delta 6 is a recessive copy-number mutant derived from Col E1.pOP1 delta 6 exists at 200-300 copies per chromosome in E. coli, while Col E1 exists at 10-15 copies per chromosome. We have investigated the molecular basis for DNA overproduction by pOP1 delta 6 by mapping the mutation to a restriction fragment of the plasmid genome, which is about 400 bp from the origin of replication. The mutation is a single base-pair alteration-a GC leads to TA transversion. The alteration changes the nucleotide sequence of two RNA elements known to be synthesized from opposite DNA strands in the same region of the plasmid genome; a small, nontranslated RNA known as RNA1 and the primer RNA required for initiation of DNA replication in vitro. The mutation is located in a GC-rich region of dyad symmetry, which precedes the termination signal for RNA1 transcription. When pOP1 delta 6 DNA is transcribed in vitro, RNA1 is not observed. Rather, several new transcripts of a larger size are observed resulting from readthrough transcription of RNA1. In conjunction with previous genetic evidence, these results indicate that RNA 1 may be a negative modulator of Col E1 DNA replication and that its secondary structure is critical to its function.