CAREER: Structural Basis of Bacterial Conjugation

希尔巴赫9733655 F因子，像所有的结合质粒一样，引导它从一种细菌转移到另一种细菌，甚至在不同物种之间。虽然F因子的遗传学已经得到了广泛的研究，但对配偶转移的结构基础却知之甚少。这位研究人员正在进行一项长期的努力，以详细描述F因子的分子结合。最初的研究将集中在三种蛋白质和质粒DNA的复合体上，这种复合体可以通过链和序列特异性促进DNA切割。在这些蛋白质中，只有质粒编码的TraI具有核溶解活性。整合宿主因子(IHF)和质粒编码的托盘是有效的Trai活性所必需的，尽管它们的作用尚不清楚。为了更好地确定托盘的活性，研究人员将通过定点突变鉴定参与DNA识别的托盘氨基酸，并将表征与托盘结合的F质粒DNA序列。IHF和托盘与DNA的结合将被评估为协同作用，以及它对DNA构象的影响。还将尝试塔板的结晶，为最终通过X射线结晶学确定结构做准备。这项研究，加上讲课和研讨会课程的教学，旨在向研究生和本科生展示使用包括结构生物学在内的跨学科研究来检查生物系统的价值。接合是一种在甚至不相关的细菌物种之间转移DNA的有效机制。因此，病原菌经常通过配偶转移获得抗生素耐药基因。在常规抗生素治疗的同时使用抑制结合的药物，可能会使抗生素更有效，并可能减少抗生素耐药性的发生率。设计这类抑制剂需要了解参与结合的蛋白质和DNA的结构和机制。拟议的活动将提供对细菌结合的一个步骤的了解，并向学生灌输对此类研究的价值和设计的欣赏。

Schildbach 9733655 F factor, like all conjugative plasmids, directs its transfer from one bacterium to another, even between species. While the genetics of F factor have been studied extensively, little is known about the structural basis of conjugal transfer. The investigator is mounting a long-term effort to describe F factor conjugation in molecular detail. The initial research will concentrate on a complex of three proteins and plasmid DNA that facilitates DNA cleavage with strand and sequence specificity. Of these proteins, only plasmid-encoded TraI possesses nucleolytic activity. Integration host factor (IHF) and plasmid-encoded TraY are required for efficient TraI activity, although their roles are unknown. To better define TraY activity, the investigator will identify TraY amino acids involved in DNA recognition through site-directed mutagenesis, and will characterize the F plasmid DNA sequence bound by TraY. The binding of IHF and TraY to DNA will be assessed for cooperativity, and for its effect on DNA conformation. Crystallization of TraY will also be attempted, in preparation for eventual structure determination by X-ray crystallography. This research, coupled with teaching in lecture and seminar courses, is intended to demonstrate to graduate and undergraduate students the value of examining biological systems using interdisciplinary studies that include structural biology. Conjugation is an efficient mechanism for transferring DNA between even unrelated bacterial species. Consequently, acquisition of antibiotic resistance genes by pathogenic bacteria frequently occurs through conjugal transfer. Drugs that inhibit conjugation, given in concert with regular antibiotic treatment, may render antibiotics more effective and may reduce the incidence of antibiotic resistance. Design of such inhibitors requires an understanding of the structures and mechanisms of the proteins and DNA involved in conjugation. The proposed activities will provide understanding of one step i n bacterial conjugation, as well as instill in students an appreciation of the value and design of such studies.