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

II型限制性内切酶修饰系统由两个基因组成，它们指定一个限制性内切酶和一个修饰甲基转移酶，在许多情况下，还有一个或多个额外的基因指定调节蛋白。这些限制性内切酶修饰系统的基因可以移动，在某些情况下自然地存在于质粒上。由于质粒本质上是可移动的元件，当这些基因进入一个完全没有甲基化的DNA的新宿主细胞时，内切酶基因的早期表达将导致宿主染色体的致命切割。一旦宿主细胞DNA被甲基化保护，甲基转移酶和核酸内切酶基因的表达比例必须改变，以有利于系统的防御功能(限制进入的噬菌体DNA)。尽管限制修饰基因对生物技术和遗传生态学很重要，但目前还没有一种限制修饰系统来详细了解其完整的调控机制。已知的是，其中几个系统由C基因家族控制，具有耐人寻味的特性。来自解淀粉芽孢杆菌和普通变形杆菌等不同细菌的C基因在交叉互补分析中功能良好。来自PvuII系统的C蛋白似乎是一个转录激活剂，但它却与转录开始的上游的DNA结合--它的结合部位与激活的启动子的表观-10六聚体重叠。该项目继续研究控制PvuII基因的调控机制。将回答三个问题。首先，C蛋白是如何激活转录的？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。这些研究将包括与一个表征芽孢杆菌蛋白质结构的X射线结晶学小组合作。其次，C.PvuII似乎也在转录启动后的一个步骤刺激内切酶基因的表达；这种刺激是否涉及内切酶基因之前的替代RNA发夹？不管是否有C.PvuII参与，这些发夹是否影响内切酶基因的表达？最后，PvuII基因在被引入新的宿主细胞后的动力学行为是什么，它是如何由调控系统的组成部分决定的？这项研究将有助于我们理解高度可传播基因的调控发展，这些基因最终可能被证明是细菌物种形成的工具。

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.