FUNCTIONAL DOMAINS OF RHIZOBIUM DCT GENES

紫花苜蓿根瘤菌和豆科根瘤菌DCT系统作为 其他细菌双组分调控系统的简单模型。每个人 有三种基因产物：膜运输蛋白(DCTA)；推定的 膜传感器(DctB)和细胞质转录激活子(DctD)。 DctD有一个用于激活转录的中心结构域，N-末端 用于抑制中心结构域的结构域，以及用于 与DCTA启动子上游的串联位点结合。在大肠杆菌中， DctD对豆科红曲霉DCTA的激活作用，但对苜蓿红曲霉DCTA的激活作用 需要集成主机因素在上游绑定 激活剂和促进剂。这项提案的主要目标是学习如何 根瘤菌将C4-二羧酸盐的存在转化为转录 DCTA在自由生活和共生状态下的激活。特定的 旨在解决以下问题。1)DctD是否协同绑定到 DCTA UAS，如果是，如何绑定？2)DCTD可以绑定到UAS中的任一站点 激活转录，还是只有DctD与启动子结合才能完成这一过程 远端部位？3)DctD的中心结构域是ATPase吗？4) 绑定ATP的能力区分活动和非活动DctD？5)是 DctD Essential的一个特定N-末端结构域残基的磷酸化 对于它的激活，或者替代的磷酸化就足够了？这些 研究包括通过聚合酶链式反应进行DNA扩增、基因克隆和遗传 DCTA启动子区域的工程；高效表达和纯化 DctD；以及ATP水解、转录的生化分析 蛋白质与DNA的启动和结合。结果将有助于 NIH的任务是了解细菌中的信号转导，这是 与毒力特性的调节有关，以及许多其他重要的 细菌活动。它们还可以通过以下方式帮助提高固氮率 根瘤菌。

The Rhizobium meliloti and Rhizobium leguminosarum dct systems serve as simple models for other bacterial two-component regulation systems. Each has three gene products: a membrane transport protein (DctA); a putative membrane sensor (DctB); and a cytoplasmic transcriptional activator (DctD). DctD has a central domain for activating transcription, an N-terminal domain for inhibiting the central domain, and a C-terminal domain for binding to tandem sites upstream of the dctA promoter. In E. coli, activation of R. leguminosarum dctA but not R. meliloti dctA by DctD requires the integration host factor to bind between upstream bound activator and promoter. The broad goal of this proposal is to learn how rhizobia transform the presence of C4-dicarboxylates into transcriptional activation of dctA, both in free-living and symbiotic states. Specific aims address the following issues. 1) Does DctD bind cooperatively to the dctA UAS, and if so, how? 2) Can DctD bound to either site in the UAS activate transcription, or is this done only by DctD bound to the promoter distal site? 3) Is the central domain of DctD an ATPase? 4) Does the ability to bind ATP distinguish active from inactive DctD? 5) Is phosphorylation of one specific N-terminal domain residue of DctD essential for its activation, or can alternative phosphorylations suffice? These studies involve DNA amplification by PCR, gene cloning, and genetic engineering of the dctA promoter region; over expression and purification of DctD; and biochemical assays for ATP hydrolysis, transcription initiation, and binding of proteins to DNA. Results will contribute to the NIH mission of understanding signal transduction in bacteria, which is relevant to the regulation of virulence properties and many other important bacterial activities. They may also aid improving nitrogen fixation by rhizobia.