Role and Mechanism of Action of C · Pvu II, a Regulatory Protein Conserved among Restriction-Modification Systems

Abstract

ABSTRACT The Pvu II restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The Pvu II system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene ( pvuIIC ) between the divergently oriented genes for the restriction endonuclease ( pvuIIR ) and modification methyltransferase ( pvuIIM ), with pvuIIC in the same orientation as and partially overlapping pvuIIR . The product of pvuIIC , C · Pvu II, was found to act in trans and to be required for expression of pvuIIR . In this study we demonstrate that premature expression of pvuIIC prevents establishment of the Pvu II genes, consistent with the model that requiring C · Pvu II for pvuIIR expression provides 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 nucleotides at their 5′ ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C · Pvu II, demonstrating that it is a sequence-specific DNA-binding protein that binds to the pvuIIC promoter and stimulates transcription of both pvuIIC and pvuIIR into a polycistronic mRNA. The apparent location of C · Pvu II binding, overlapping the −10 promoter hexamer and the pvuIICR transcriptional starting points, is highly unusual for transcriptional activators.