Replication control mutations of plasmid R6-5 and their effects on interactions of the RNA-I control element with its target.

Nine high copy number mutations of plasmid R6-5, representing five phenotypically distinct groups, have been identified by DNA sequencing. In each mutant plasmid examined, a single nucleotide change was found. The effects of the mutations on possible gene products, and DNA-RNA secondary structure, were analyzed and compared with the observed phenotypes. The results of this study exclude the possibility that the primary plasmid replication control element, the product of the copA gene, is a polypeptide, and they are consistent with a model of plasmid replication control by the copA product which has the following features: (i) RNA-I, a short untranslated RNA molecule, is the product of the copA gene and regulates the frequency of initiation of plasmid replication, (ii) the hexanucleotide single-strand loop of the major hairpin of RNA-I is its active site, (iii) this active site functions by base pair interactions with its "target," its DNA template strand, or its complementary sequence on RNA-II, a transcript of opposite polarity that is the message of the repA gene, and (iv) the sequence and size of the loop, and the stability of the stem of the hairpin, are all critical factors that govern the functioning of RNA-I.