Identification of the minimal replicon and the origin of replication of the crenarchaeal plasmid pRN1

We have determined the minimal replicon of the crenarchaeal plasmid pRN1. It consists of 3097 base pairs amounting to 58% of the genome of pRN1. The minimal replicon comprises replication operon orf56/orf904 coding for a transcriptional repressor and the replication protein of pRN1. An upstream region of 64 bp that contains the promoter of the replication operon is essential as well as 166 bp of sequence downstream of the orf904 gene. This region contains a putative transcriptional terminator and a 100 nucleotides long stem–loop structure. Only the latter structure was shown to be required for replication. In addition replication was sustained when the stem–loop was displaced to another part of the pRN1 sequence. By mutational analysis we also find that the integrity of the stem–loop structure is required to maintain the replication of pRN1-derived constructs. As similar stem–loop structures are also present in other members of the pRN family, we suggest that this conserved structural element could be the origin of replication for the pRN plasmids. Further bioinformatic analysis revealed that the domain structure of the replication protein and the presence of a similar stem–loop structure as the putative replication origin are also found in several bacteriophages.