Regulation of restriction-modification genes expression

描述（由申请人提供）：本研究将主要在俄罗斯普希诺的俄罗斯科学院微生物生物化学和生理学研究所（IBPM）与亚历山大索洛宁博士合作进行。长期目标是了解II型限制性内切酶和甲基转移酶基因的限制性修饰（R-M）系统的时间协调表达。这些系统很普遍;它们对细菌基因组进化有重要贡献，并且作为基因工程工具的来源具有实际意义。美国和俄罗斯团队的互补优势将结合起来，在分子水平上研究确保两种不同II型R-M系统中R-M基因表达受调控的机制。 具体目标1. EcoRV的调节C蛋白转录调节的分子机制。通过遗传学和生物化学实验确定了EcoRV.R（限制性内切酶）基因转录激活和EcoRV.M（甲基转移酶）基因转录抑制的分子机制，这些分子机制是由专门的DNA结合控制因子EcoRV. C蛋白（EcoRV系统编码的第三种蛋白质）激活的。还将进行EcoRV. C的结构分析及其与DNA的相互作用。比较分析的结果有望揭示许多R-M系统编码的同源C蛋白的转录调控机制。 具体目标2。SsoII的转录调控。SsoII.M甲基化酶含有识别R-M调节区中的操纵位点的DNA结合结构域。遗传和生化实验将确定ssoII.M基因转录的自阻遏和ssoII.R基因表达的激活的分子机制。 这些实验的结果将描述新的监管机制，导致细菌基因表达的转录水平的时间调节。拟议中的工作策略的转录调控的遗传移动的R-M系统将补充实验，旨在表征宿主转录调控的分子机制所采用的噬菌体，这是在父母NIH补助金的核心。

DESCRIPTION (provided by applicant): 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.