ColEI-Type Replicons

The replicon of the ColEl plasmid of Escherichia coli is the basis for many gene-cloning and gene-expression vectors that are commonly used in current molecular biology (see Parts 2 and 28). In contrast to the replication of iteron-containing plasmids, ColE1 replication proceeds without a plasmid-encoded replication initiation protein and instead utilizes an RNA species in initiation and RNA-RNA interactions to achieve copy number control (see Fig. 2B) (46).

ColE1 uses an extensive RNA primer for leading-strand synthesis. The RNAII preprimer is transcribed from the RNAII promoter by host RNA polymerase. RNAII forms a persistent RNA-DNA hybrid at the plasmid origin of replication. This hybrid is cleaved by RNase H and the resulting free 3'OH group on the cleaved RNAII acts as a primer for continuous leading-strand synthesis, catalyzed by host DNA polymerase I.

ColE1 regulates its copy number with a short RNA countertranscript, RNAI. This species is expressed constitutively from the strong RNAI promoter, is nontranslated, and is fully complementary to part of RNAII. The interaction of RNAI with RNAII results in an RNAII configuration that