Molecular biology of the pRN1 plasmid from Sulfolobus islandicus

Abstract

The pRN1 plasmid is a rather small multicopy plasmid which was isolated from a Sulfolobus islandicus strain in 1993 by Wolfram Zillig and co-workers. Sequence analysis of the genome sequence suggested that three conserved genes are important for plasmid replication. These genes code for two sequence-specific DNA-binding proteins (ORF56 and ORF80) and for a large multifunctional replication protein (ORF904). The protein ORF904 has primase, DNA polymerase and helicase activity. Remarkably, the primase activity is highly sequence specific, and primers are only efficiently synthesized on templates with the motif GTG. This protein could initiate the plasmid replication by melting the double-stranded DNA at the origin of replication and by synthesizing the first primers at the replication bubble. The protein ORF56 is a repressor, and combined biochemical and genetic evidence shows that this protein is involved in regulating the copy number of the plasmid. The function of the third conserved protein, ORF80, is still mysterious. Although this protein is highly conserved, it is not essential for replication, since shuttle vectors with a deleted orf80 gene are still able to replicate in Sulfolobus. Interestingly, plasmids lacking the orf80 gene display reduced plasmid retention under non-selective conditions, raising the possibility that ORF80 is involved in plasmid partitioning or has an accessory role in plasmid replication.