Transciptional Activation by Rhizobium meliloti DCTD

HOOVER 96-30454本项目的长期目标是了解β 54 RNA聚合酶全酶β 54 ~n细菌转录激活的相关机制。尽管在现象学水平上已经知道了很多，但关于这一过程中所涉及的机制的许多细节还需要阐明。E β 4与启动子结合形成封闭复合物，不能启动转录。结合在启动子上游的激活剂必须通过DNA成环与E β 4进行有效接触并水解ATP以激活转录。激活剂和E β 4之间的相互作用是短暂的，并且对E β 4和任何依赖于C β的激活剂之间的特定接触位点一无所知。识别这些接触位点对于剖析E（54）的转录激活机制至关重要，本项目的目标是识别这些接触位点。来自苜蓿根瘤菌的（54 -依赖性激活剂DctD）（C4二羧酸转运蛋白D）将用于这些研究。DctD可与RNA聚合酶的β 4和β，B亚基交联，表明DctD可与β 4的这些亚基结合以激活转录。已经产生了不能激活转录的DctD）突变体的集合，并且这些突变体中的一些可能在与E（54.将对抑制dctD突变体的ntrA（编码（54））和rpo B（编码RNA聚合酶的B亚基）的等位基因进行搜索。将纯化这些抑制子的基因产物，并在体外转录测定中检查它们与DctD突变体一起发挥功能的能力，以及在交联测定中检查它们与DctD突变体相互作用的能力。第二个研究目标是分离（54个在与DctD的蛋白质-蛋白质相互作用中有缺陷的突变体：）。预期这样的β 4突变体允许β 4与启动子结合以形成封闭复合物，但不允许这些封闭复合物-开放复合物的异构化，所述开放复合物是转录生产性的。基于E β 4抑制噬菌体P22 ant基因转录的能力，已经开发了一种遗传筛选，并将用于分离具有这些特性的突变体。将检查从该筛选分离的突变体识别启动子序列、结合核心RNA聚合酶和在交联测定中与DctD相互作用的能力。这些实验的结果将有助于确定DctD和E之间的接触位点。 研究β 54 -依赖性激活剂如何发挥作用是至关重要的，因为它将有助于我们了解细菌中有多少重要的代谢过程受到控制。在细菌中，β 4-依赖性基因的存在是广泛的，并且这些基因的产物参与诸如固氮和代谢、环境污染物的降解以及植物和人类病原体中的毒力因子的调节等多种过程。了解细菌如何控制这些代谢过程对于在农业，环境保护和医学中控制和利用这些生物体至关重要。

HOOVER 96-30454 The long term goal of this project is to understand be mechanisms involved in transcriptional activation with (54 RNA polymerase holoenzyme (E(54) ~n bacteria Although much is known at a phenomenological level, many details concerning the mechanisms involved in this process need to be elucidated. E(54 binds to the promoter to form a closed complex that is unable to initiate transcription. The activator, which binds upstream of the promoter, must make productive contact with E(54 through DNA looping and hydrolyze ATP in order to activate transcription. Interactions between the activator and E(54 are transient, and nothing is known about specific contact sites between E(54 and any c~ dependent activator. Identifying these contact sites is crucial for dissecting the mechanisms of transcriptional activation with E(54, and the goal of this project is to identify these contact sites. The (54 -dependent activator DctD) (C4 dicarboxylic acid transport protein D) from Rhizobium meliloti will be used for these studies. DctD can be crosslinked to (54 and the ,B subunits of RNA polymerase, suggesting that DctD may engage these subunits of E(54 to activate transcription. A collection of DctD) mutants that fail to activate transcription bas been generated, and some of these mutants are likely to be defective in interactions with E(54. A search will be made for alleles of ntrA (encodes (54) and rpoB (encodes the B subunit of RNA polymerase) that suppress dctD mutants. Gene products of these suppressors u ill be purified and examined for their ability to function with the DctD mutants in an In vitro transcription assay, as well as their ability to interact with DctD mutants in the crosslinking assay. A second research goal is to isolate (54 mutants that are defective in protein-protein interactions with DctD:). Such (54 mutants are expected to allow E(54 to bind to the promoter to form a closed complex, but will not allow the isomerization of these closed complexes ~ open complexes that are tr anscriptionally productive. A genetic screen based on the ability of E(54 to repress transcription from the phage P22 ant gene has been developed, and will be used to isolate (54 mutants with these properties. The (54 mutants isolated from this screen will be examined for their ability to recognize promoter sequences, bind core RNA polymerase, and interact with DctD in the crosslinking assay The results of these experiments will help define the contact sites between DctD and E(54. Examination of how (54 -dependent activators function is crucial, as it will contribute to our understanding of how many important metabolic processes are controlled in bacteria. The occurrence of (54 -dependent genes in bacteria is widespread, and the products of these genes are involved in such diverse processes as nitrogen fixation and metabolism, degradation of environmental pollutants, and regulation of virulence factors in plant and human pathogens. Understanding how bacteria control such metabolic processes is essential for the control and exploitation of these organisms in agriculture, environmental protection, and medicine.