Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA.

Abstract

Transcription of ColE1 DNA by RNA polymerase in vitro starts at two sites in a region required for maintenance of the plasmid. Certain transcripts that start at one of the sites can be cleaved by RNase H and then act as primers for DNA replication. Transcription from the other site produces a RNA approximately 108 nucleotides long (species I or RNA I). Transcripts analogous to the primer and RNA I of ColE1 are produced when p15A or small derivatives of two other ColE1-compatible plasmids, CloDF13 and RSF1030, are used as template. If purified RNA I is added to the transcription reaction containing RNase H, formation of primer is inhibited. Each RNA I can inhibit primer formation by the plasmid that specifies it but has no effect on primer formation by heterologous templates. Thus, the inhibition of primer formation by RNA I is incompatibility specific. Because RNA I does not inhibit initiation or propagation of transcription or the processing of preformed precursors, the step that is sensitive to inhibition is probably formation of the hybrid between the primer precursor and the template. This hybrid is the required substrate for RNase H. Experiments with recombinant plasmids show the region that determines the specificity of response to RNA I to be greater than 300 base pairs upstream of the origin of DNA replication.