Role and mechanism of action of C center dot PvuII, a regulatory protein conserved among restriction-modification systems

The PvuII restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are in dependently active proteins. The PvuII system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by e xpression of the methyltransferase gene alone so that the new; host's DNA i s protected before endonuclease activity appears. Previous studies have ide ntified a regulatory gene (pvuIIC) between the divergently oriented genes f or the restriction endonuclease (pvuIIR) and modification methyltransferase (pvuIIM), with pvuIIC in the same orientation as and partially overlapping pvuIIR, The product of pvuIIC, C . PvuII, was found to act in trans and to be required for expression of pvuIIR. In this study we demonstrate that pr emature expression of pvuIIC prevents establishment of the PvuII genes, con sistent with the model that requiring C . PvuII for pvuIIR expression provi des a timing delay essential for protection of the new host's DNA, We find that the opposing pvuIIC and pvuIIM transcripts overlap by over 60 nucleoti des at their 5' ends, raising the possibility that their hybridization migh t play a regulatory role. We furthermore characterize the action of C . Pvu II, demonstrating that it is a sequence-specific DNA-binding protein that b inds to the pvuIIC promoter and stimulates transcription of both pvuIIC and pvuIIR into a polycistronic mRNA. The apparent location of C . PvuII bindi ng, overlapping the - 10 promoter hexamer and the pvuIICR transcriptional s tarting points, is highly unusual for transcriptional activators.