RUI: Biochemical and Genetic Characterization of Membrane- Associated VirB-Protein COmplexes in Agrobacterium tumefaciens

RUI：根癌农杆菌膜相关 VirB 蛋白复合物的生化和遗传表征

• 批准号: 9506144
• 负责人: Lois Banta
• 金额: $ 31万
• 依托单位: Haverford College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-15 至 1999-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9506144&HistoricalAwards=false
• 关键词: RUI; Biochemical; Genetic; Characterization; Membrane

; R o o t E n t r y F g 5 @ C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l :z 5 :z 5 F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT marcia steinberg marcia steinberg @ _ 5 @ e = e { n " " " " " " " L L L L L d n L C x | | | | | | | # ı e V T 4 " | | | | | | " " | x | | | | " | " | 6 " " " " | | 4 | 9506144 Banta The plant pathogen Agrobacterium tumefaciens causes a disease in which crown gall tumors form on susceptible plants. The disease results from a complex interaction that is initiated when the bacterium binds to the plant cell surface at a wound site. The bacterium has the unusual ability to transfer a fragment of DNA (the T DNA) into a host plant cell and deliver the DNA to the nucleus, where it becomes part of the host plant's genome. Extensive molecular characterization over the past several years has helped elucidate the process of T DNA production within the bacterium, as well as the mechanism by which a molecule of bacterial origin is directed to a specific compartment in a eukaryotic cell. One of the fun damental aspects of this plant pathogen interaction that remains shrouded in mystery is the actual transport process mediating export of the T DNA from the bacterium and delivery into the plant cell. A multiprotein channel or pore complex has been hypothesized to allow passage of the T DNA (and its associated proteins) through the two membranes that surround the bacterium. Several candidate proteins have been identified that could make up such a transport apparatus; these proteins are encoded by the virB operon on the large tumor inducing (T) plasmid that carries many of the virulence associated genes (as well as the T DNA itself). Recent results from a number of labs including our own have shed some light on the localization and other characteristics of many of the VirB proteins; these observations support a model in which several VirB proteins interact to form a T DNA transport pore, while others many function as chaperones and/or sources of energy for the translocation event. The proposed studies are designed to determine which VirB proteins interact to form oligomeric complexes. Genetic analyses will take advantage of point mutations in the virB10 gene that result in a loss of tumor formation; potentially interacting species will be identified by screening for compensatory changes in a second virB gene that can suppress the avirulent phenotype of the original mutant. A second genetic approach will utilize the "dihybrid" strategy, in which proteins or protein fragments are tested for their ability to bring together two domains of a yeast transcriptional activator such as the GAL4 protein. These genetic studies will be complemented by biochemical experiments designed to explore the composition of VirB protein containing aggregates. Together, these studies should provide some insight into the mechanism by which a large polar molecule such as the T DNA and associated proteins is transported across the two apolar membranes that enclose the Agrobacterium cell. Molecular dissection of the structure and function of the putative transfer apparatus will, in turn, help elucidate the processes and interactions mediating assembly of what is thought to be an elaborate membrane associated complex. %%% The soil bacterium Agrobacterium tumefaciens infects susceptible plants, resulting in unregulated cell division and crown gall tumor formation. This disease can be thought of as "plant cancer" in that, as with human tumors, cells are multiplying at an inappropriate time and/or place. Infection by Agrobacterium involves the movement of a piece of DNA (the T DNA) from the bacterium into the host plant cell. This unusual interaction is the only known naturally occurring example of DNA transfer from one kingdom (bacterial) to another (plant). We are especially interested in the mechanism by which a large water loving molecule of DNA is able to cross the oily membrane that surrounds the bacterial cell. T DNA processing and transfer are mediated by a number of bacterial virulence (Vir) proteins. One group of Vir proteins, the VirB proteins, are believed to play a direct role in allowing the T DNA to exit the bacterial cell, most likely by interacting to form an opening in the bacterial membrane through which the T DNA is transferred. We plan to test this hypothesis, using both genetic and biochemical approaches, by studying defective VirB proteins that do not permit T DNA movement, and by isolating complexes of VirB proteins. *** ; @ ....()()))()() ;

; r o o o o t e n t r y f g 5 @ c o m p o b j b j b j b j b j b d d o c u c c u c u c c u c b j e c c t p o o l：z 5：z 5 f microsoft Word 6.0文档文档mworddoc word.document.document.6;哦 +'0 $ h l d h r：\ wwuser \ template \ normal.dot marcia steinberg marcia steinberg @ _ 5 @ e = e = e = e = e {n“”“”“”“” l l l l l l l l l l l l l l l l l c x | | | | | | | | | | | | | | | | | | | | | | | | “” | X | | | | “ |” | 6“”“” | 4 | 9506144 BANTA植物病原体农杆菌Tumefaciens引起了一种疾病，其中易感植物形成牙冠胆肿瘤。该疾病是由于细菌与伤口部位的植物细胞表面结合时引发的复杂相互作用而引起的。细菌具有将DNA（T DNA）片段转移到宿主植物细胞中并将DNA传递到细胞核中的异常能力，并将其变成宿主植物基因组的一部分。在过去的几年中，广泛的分子表征有助于阐明了细菌内的T DNA产生过程，以及细菌来源分子的机制针对真核生物细胞中的特定隔室。这种植物病原体相互作用的有趣的典型方面之一是神秘的笼罩在神秘中的实际运输过程，它介导了从细菌中介导的T DNA出口并递送到植物细胞中。已经假设了多蛋白通道或孔复合物，以使T DNA（及其相关蛋白）通过围绕细菌的两种机制通过。已经确定了几种可以构成这种运输机构的候选蛋白质。这些蛋白质是由ViRB歌剧编码的，该蛋白具有许多毒力相关基因（以及T DNA本身）的大肿瘤（T）质粒（T）质粒。许多实验室（包括我们自己的实验室）的最新结果已经阐明了许多VIRB蛋白的本地化和其他特征。这些观察结果支持了一个模型，其中几种VIRB蛋白相互作用以形成T DNA传输孔，而其他观测则是易位事件的伴侣和/或能源的许多作用。拟议的研究旨在确定哪种VIRB蛋白相互作用以形成寡聚复合物。遗传分析将利用VIRB10基因中的点突变，从而导致肿瘤形成损失。通过筛选第二个VIRB基因的代码变化，可以识别潜在的相互作用物种，该基因可以抑制原始突变体的无毒表型。第二种遗传方法将利用“二杂交”策略，其中对蛋白质或蛋白质片段进行了测试，其能力将酵母转录激活剂的两个结构域（例如GAL4蛋白）汇总在一起。这些遗传研究将通过旨在探索含有聚集体的VIRB蛋白组成的生化实验完成。总之，这些研究应提供一些对大型极性分子（例如T DNA和相关蛋白）的机制的洞察力，这些机制跨越了包围农业细胞的两种极性机制。推定转移设备的结构和功能的分子解剖反过来，将有助于阐明介导的过程和相互作用，介导了被认为是精致的膜相关复合物的组装。 %% %%土壤细菌农杆菌感染易感植物，导致细胞分裂和冠状胆囊形成。该疾病可以被认为是“植物性癌”，因为与人类肿瘤一样，细胞在不适当的时间和/或地方繁殖。农杆菌感染涉及一块DNA（T DNA）从细菌转移到宿主植物细胞的运动。这种不寻常的相互作用是唯一已知的自然发生的DNA从一个王国（细菌）转移到另一个植物（植物）的例子。我们对大型DNA的大量水分子能够越过细菌细胞周围的油性膜的机制特别感兴趣。 T DNA加工和转移是由许多细菌毒力（VIR）蛋白介导的。据信，一组VIRB蛋白（VIRB蛋白）在允许T DNA退出细菌细胞中起着直接的作用，这很可能是通过相互作用以在细菌膜中形成开口的TNA中的开口。我们计划使用遗传和生化方法，通过研究不允许DNA运动的有缺陷的VIRB蛋白，并通过分离ViRB蛋白的复合物来检验该假设。 ***; @ ...（）（）））（）;