Conditional high copy number ColE1 mutants: resistance to RNA1 inhibition in vivo and in vitro.EMBO J 1988; 7(10):3289-97EJ
We describe three independently isolated copy number mutants of a plasmid ColE1 derivative which undergo temperature- and growth-phase-dependent DNA amplification in Escherichia coli. These mutants have single base-pair alterations in a highly localized region of the plasmid genome encoding the replication primer RNA. The mutations map immediately upstream of the RNA1 transcript, altering the sequence between conserved elements of the RNA1 promoter. These mutants have 2- to 4-fold increased copy number relative to wild-type plasmids in exponential growth at 37 degrees C but undergo 20-fold amplification of copy number relative to wild-type when cells enter stationary phase. Cells containing these plasmids grow with normal kinetics at 37 degrees C but grow poorly at 42 degrees C. The poor growth is associated with high-level plasmid amplification. Both the temperature and growth phase plasmid DNA amplification are suppressed if the ColE1 rop gene product is provided in trans from a compatible plasmid. Analysis of steady-state RNA1 levels indicates that DNA amplification occurs in the presence of RNA1 made by the mutant plasmid. Thus, the DNA amplification of the copy mutant is not due to an inability to synthesize RNA1. Using an in vitro transcription system containing RNase H, we show that mutant primer processing by RNase H is resistant to levels of the replication initiation inhibitor RNA1 that inhibit wild-type primer processing. The defect in inhibition appears not to be at the level of association of RNA1 with nascent primer. These results indicate that mutant plasmid amplification is due to the ability of its primer precursor transcripts to serve as substrates for RNase H despite the presence of RNA1.