This research will be done primarily in the Institute of Biochemistry and Physiology of Microorganisms (IBPM), Russian Academy of Sciences, Puschino, Russia, in collaboration with Dr. Alexander Solonin. The long-term goal is to understand the temporally coordinated expression of restriction endonuclease and methyltransferase genes of Type II restriction-modification (R-M) systems. These systems are widespread; they contribute significantly to bacterial genome evolution and are practically significant as a source of tools for genetic engineering. The complementary strengths of the US and Russian groups will be combined to study, at the molecular level, mechanisms that ensure regulated R-M gene expression in two different Type II R-M systems. Specific aim 1. Regulation of EcoRV the molecular mechanism of transcription regulation by C-proteins. Genetic and biochemical experiments are proposed to define the molecular mechanisms of activation of the ecoRV.R (restriction endonuclease) gene transcription and repression of the ecoRV.M (methyltransferase) gene transcription by specialized DNA binding control factor, the EcoRV.C protein, the third protein encoded by the EcoRV system. Structural analysis of EcoRV.C and its interaction with DNA will also be performed. The results of the comparative analyses are expected to shed light on transcription regulation mechanism used by homologous C proteins which are encoded by numerous R-M systems. Specific aim 2. Transcription regulation of SsoII. The SsoII.M methylase contains a DNA binding domain that recognizes an operator site in the R-M regulatory region. Genetic and biochemical experiments will define the molecular mechanism of autorepression of the ssoII.M gene transcription by ssoII.M and activation of ssoII.R gene expression. The results of these experiments will describe novel regulatory mechanisms that lead to temporal regulation of bacterial gene expression at the level of transcription. The proposed work on strategies of transcription regulation by genetically mobile R-M systems will complement experiments aimed at characterizing molecular mechanisms of host transcription regulation employed by bacteriophages, which are at the core of the parent NIH grant.