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

不寻常的移动调节盒的作用机制：限制性修饰系统的 C 基因

• 批准号: 9904523
• 负责人: Robert Blumenthal
• 金额: $ 35万
• 依托单位: University of Toledo Health Science Campus
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9904523&HistoricalAwards=false
• 关键词: Mechanism; Action; Unusual; Mobile; Regulatory

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.

II型限制性修饰系统包含指定限制性核酸内切酶和修饰甲基转移酶的两个基因，以及在许多情况下指定调节蛋白的一个或多个附加基因。 这些限制性修饰系统的基因可以是可移动的，在某些情况下天然存在于质粒上。 因为质粒本质上是移动元件，当这些基因移动到具有完全未甲基化DNA的新宿主细胞中时，核酸内切酶基因的早期表达将导致宿主染色体的致命裂解。 一旦宿主细胞 DNA 受到甲基化保护，甲基转移酶和核酸内切酶基因的表达比例就必须改变，以有利于系统的防御功能（限制噬菌体 DNA 的进入）。 尽管限制性修饰基因对生物技术和遗传生态学很重要，但还没有一种限制性修饰系统可以详细了解其完整的调控机制。 已知其中一些系统是由调节因子家族（C 基因）控制的，具有有趣的特性。 来自解淀粉芽孢杆菌和普通变形杆菌等不同细菌的 C 基因在交叉互补测定中功能良好。 来自 PvuII 系统的 C 蛋白似乎是一种转录激活剂，但与转录起始上游的 DNA 结合——其结合位点与激活启动子的明显 -10 六聚体重叠。 该项目继续研究控制 PvuII 基因的调控机制。 将解决三个问题。 首先，C蛋白如何激活转录？ 为了解决这个问题，并了解 C 蛋白的广泛宿主范围，将与芽孢杆菌和变形杆菌蛋白进行比较研究。 这些研究将包括与 X 射线晶体学小组合作，表征芽孢杆菌蛋白的结构。 其次，C.PvuII 似乎还在转录起始后的步骤中刺激核酸内切酶基因的表达；这种刺激是否涉及核酸内切酶基因之前的替代 RNA 发夹？ 无论是否涉及 C.PvuII，这些发夹是否会影响核酸内切酶基因的表达？ 最后，PvuII 基因引入新宿主细胞后的动力学行为是什么？它是如何由调节系统的组成部分决定的？ 这项研究将有助于我们了解高度可传递基因的调控发展，这些基因可能最终被证明有助于细菌物种形成。