An antisense RNA plays a central role in the replication control of a repC plasmid.

The widespread replicons of repABC and repC families from alpha-proteobacteria share high similarity in their replication initiator proteins (RepC). Here we describe the minimal region required for stable replication of a member of the repC family, the low copy-number plasmid pRmeGR4a from Sinorizobium meliloti GR4. This region contains only two genes: one encoding the initiator protein RepC (46.8 kDa) and other, an antisense RNA (67 nt). Mapping of transcriptional start sites and promoter regions of both genes showed that the antisense RNA is nested within the repC mRNA leader. The constitutively expressed countertranscribed RNA (ctRNA) forms a single stem-loop structure that acts as an intrinsic rho-independent terminator. The ctRNA is a strong trans-incompatibility factor and negative regulator of repC expression. Based on structural and functional similarities between members of the repC and repABC families we propose a model of their evolutionary relationship.