Fig. 1. ColE1-like plasmid copy number regulation by antisense RNA.

a. Original pMB1 plasmid. b. pUC19 origin of replication present in pGFPuv. ColE1 plasmid replication is regulated by sequence elements known as the plasmid origin of replication (ori). Replication starts with the transcription of an RNA pre-primer. This RNA pre-primer (RNAII) forms a stable hybrid at its 3’ end with ori DNA (R-loop). Processing of this R-loop RNA by RNaseH generates a free 3’–OH end that is extended by DNA polymerase I (Pol I), initiating leading-strand synthesis (Itoh and Tomizawa, 1980). Replication initiation is negatively regulated by an antisense RNA mechanism that maintains copy number constant for a given physiological condition (Brantl, 2014; Camps, 2010). The 108 bp antisense RNA (RNAI) is transcribed from a strong promoter located downstream and in the opposite orientation from the promoter for RNAII. RNAI is short-lived and forms a stable hybrid with the pre-primer RNA, blocking downstream R-loop formation by irreversibly altering the folding of the pre-primer (Cesareni et al., 1991; Polisky, 1988). In the original pMB1 origin of replication, the hybridization between RNAI and RNAII is facilitated and stabilized by a small dimeric protein, Rom (for RNA one modulator) (Lacatena et al., 1984; Tomizawa and Som, 1984). pUC19 is a pMB1-derivative lacking rom and harboring a C→T mutation in the ori, immediately downstream of the RNAI promoter, leading to a G→A substitution in the RNAII. This mutation appears to alter the secondary structure of pUC19’s RNAII, promoting normal folding of the pre-primer. As a result, pUC19 exhibits a several-fold increase in plasmid copy number relative to its parental pMB1 (Lin-Chao et al., 1992).