Regulation of restriction-modification genes expression

描述(申请人提供)：这项研究将主要在俄罗斯科学院生物化学和微生物生理学研究所(IBPM)与亚历山大·索洛宁博士合作完成。长期目标是了解II型限制性内切酶和甲基转移酶基因在时间上的协调表达。这些系统分布广泛；它们对细菌基因组进化做出了重大贡献，并且作为基因工程工具的来源实际上具有重要意义。美国和俄罗斯团队的互补优势将结合在一起，在分子水平上研究确保两个不同类型R-M系统中受调控的R-M基因表达的机制。 具体目的1.对EcoRV的调控C蛋白转录调控的分子机制。通过遗传学和生物化学实验来确定EcoRV.R(限制性内切酶)基因转录的激活和EcoRV.M(甲基转移酶)基因转录被特殊的DNA结合控制因子EcoRV.C蛋白(EcoRV.C蛋白)抑制的分子机制。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阐明许多R-M系统编码的同源C蛋白所使用的转录调控机制。 特定目的2.SsoII的转录调控。SsoII M甲基酶包含一个DNA结合域，它识别R-M调节区中的操纵子位点。遗传和生化实验将确定ssoII M自身抑制ssoII M基因转录和激活ssoII R基因表达的分子机制。 这些实验的结果将描述新的调控机制，导致在转录水平上对细菌基因表达的时间调控。关于基因移动R-M系统转录调控策略的拟议工作将补充旨在表征噬菌体宿主转录调控分子机制的实验，噬菌体是父NIH拨款的核心。

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.