CAREER: Structural Basis of Bacterial Conjugation

Schildbach因子，像所有的共轭质粒一样，引导它从一个细菌转移到另一个细菌，甚至在物种之间。虽然F因子的遗传学已被广泛研究，但对配偶转移的结构基础知之甚少。研究者正在进行长期的努力来描述F因子结合的分子细节。最初的研究将集中在三种蛋白质和质粒DNA的复合体上，这种复合体可以促进DNA的链和序列特异性切割。在这些蛋白中，只有质粒编码的TraI具有溶核活性。整合宿主因子（IHF）和质粒编码托盘是有效的TraI活性所必需的，尽管它们的作用尚不清楚。为了更好地定义TraY活性，研究者将通过位点定向诱变鉴定参与DNA识别的TraY氨基酸，并将表征与TraY结合的F质粒DNA序列。将评估IHF和TraY与DNA的结合的协同性及其对DNA构象的影响。还将尝试托盘的结晶，为用x射线晶体学确定最终的结构做准备。这项研究，加上讲座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研究生物系统的价值。偶联是一种有效的转移DNA的机制，即使是不相关的细菌物种。因此，病原菌获得抗生素耐药基因往往是通过配偶转移。抑制结合的药物与常规抗生素治疗配合使用，可使抗生素更有效，并可减少抗生素耐药性的发生率。设计这样的抑制剂需要了解结构和机制的蛋白质和DNA参与缀合。拟议的活动将提供一个步骤的理解细菌结合，以及在学生灌输的价值和这种研究的设计欣赏。

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.