Mechanism of Action of an Unusual Mobile Regulatory Cassette: The C Genes of Restriction-Modification Systems

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Type II restriction-modification systems comprise two genes that specify a restriction endonuclease and a modification methyltransferase, and in many cases one or more additional genes that specify regulatory proteins. The genes for these restriction-modification systems can be mobile, in some cases naturally residing on plasmids. Because plasmids are essentially mobile elements, when these genes move into a new host cell that has completely unmethylated DNA, early expression of the endonuclease gene would lead to lethal cleavage of the host chromosome. Once the host cell DNA has been protected by methylation, the expression ratio of the methyltransferase and endonuclease genes must change to favor the defensive function of the system (restriction of incoming bacteriophage DNA). Despite the importance of restriction-modification genes to biotechnology and genetic ecology, there is no restriction-modification system for which the full complement of regulatory mechanisms is understood in detail. Several of these systems are known to be controlled by a family of regulators, the C genes, with intriguing properties. C genes from bacteria as different as Bacillus amyloliquefaciens and Proteus vulgaris function well in cross-complementation assays. The C protein from the PvuII system appears to be a transcriptional activator and yet binds to the DNA just upstream of the start of transcription -- its binding site overlaps the apparent -10 hexamer of the activated promoter. This project continues the study of the regulatory mechanisms that control the PvuII genes. Three questions will be addressed. First how do the C proteins activate transcription? To address this question, and to understand the broad host range of the C proteins, comparative studies will be carried out with the Bacillus and Proteus proteins. These studies will include collaboration with an x-ray crystallography group characterizing the structure of the Bacillus protein. Second, C.PvuII also appears to stimulate expression of the endonuclease gene at a step subsequent to transcription initiation; does this stimulation involve the alternative RNA hairpins that precede the endonuclease gene? Whether or not C.PvuII is involved, do these hairpins affect expression of the endonuclease gene? Finally, what is the kinetic behavior of the PvuII genes following introduction into new host cells, and how is it determined by the components of the regulatory system? This research will contribute to our understanding of development of regulation of highly transmissable genes that may ultimately prove to be instrumental in bacterial speciation.