Transcriptional Activation by Rhizobium meliloti DCTD

胡佛根瘤菌（Hoover Rhizobium meliloti） DCTD激活dctA（编码c4 -二羧酸转运蛋白）的转录，属于一个依赖于（54）的激活因子家族，可能通过共同的机制起作用。依赖Sigma54的基因编码的产物参与了多种重要的代谢过程，包括氮同化、固氮、c4 -二羧酸运输、甲苯降解、氢代谢和pilin形成。本实验室的长期目标是了解（54）依赖性激活因子转录激活的分子机制。研究(54依赖的激活因子如DCTD如何促进转录激活将有助于理解细菌基因调控和生理。54 -RNA聚合酶全酶（E(54)）的转录机制与主要形式的RNA聚合酶全酶（E(70)）有几个显著的不同。例如，激活剂对ATP的水解与E（54）耦合形成开放络合物，而在E（70）转录的众多基因中，没有这种对ATP需求的例子。为了实现这一长期目标，以下短期目标将在本研究中解决。首先，在激活转录方面有缺陷的DCTD突变体将会产生并被表征。在这些实验中，将使用截断形式的DCTD（称为DCTDL143），该DCTDL143可组成性地激活转录并水解ATP。突变体DCTDL143蛋白将被纯化并分析其在体外激活转录、水解ATP、结合DNA和与E（54）相互作用的能力。DCTD可以与RNA聚合酶（54）和（亚基）交联，DCTDL143突变体与E（54）亚基之间的相互作用将通过这种交联试验进行检测。这些生化和遗传方法对于解剖转录激活所需的蛋白质-蛋白质相互作用将是有价值的。其次，对DCTDL143的ATP酶活性的研究将扩展到包括检查潜在的抑制剂，确定温度和pH最优，以及确定ATP的解离常数。光交联ATP（或ATP类似物）与DCTD将用于识别参与ATP结合的蛋白质区域。检测DCTDL143和DCTDLl43突变体的ATP结合和水解可能会对ATP水解如何与转录激活耦合产生深刻的见解。总之，这些数据将为DCTD如何激活来自dctA启动子的转录提供有用的信息。尽管在原核生物和真核生物中已经发现了大量的转录激活因子，但人们对这些蛋白质促进转录激活的方式知之甚少。本实验室的长期目标是表征细菌中一类激活剂(（54 -依赖）的转录激活的分子机制。其中一种活化剂，乳酸菌的DCTD将被研究。缺乏激活转录能力的突变激活因子可以通过随机和局部诱变产生。这些突变蛋白将被检测其激活转录、结合和水解ATP以及与E（54）相互作用的能力。对截断形式的DCTD的ATP酶活性的研究将扩展到包括检查潜在的抑制剂，确定ATP水解的最佳温度和pH值，确定MgATP的解离常数，并确定参与MgATP结合的区域。这项工作将有助于增加我们对细胞如何激活那些适合特定环境的基因的理解。＊＊＊

9506333 Hoover Rhizobium meliloti DCTD activates transcription from dctA (which encodes a C4-dicarboxylate transport protein) and belongs to a family of (54-dependent activators that probably operate by a common mechanism. Sigma54 -dependent genes encode products that are involved in important and diverse metabolic processes, including nitrogen assimilation, nitrogen fixation, C4-dicarboxylate acid transport, toluene degradation, hydrogen metabolism, and pilin formation. The long term goal of this lab is to understand the molecular mechanisms involved in transcriptional activation by (54 -dependent activators. Examining how (54-dependent activators like DCTD facilitate transcriptional activation will contribute greatly to the understanding of bacterial gene regulation and physiology. The transcription mechanism for (54 -RNA polymerase holoenzyme (E(54) differs significantly from that of the major form of RNA polymerase holoenzyme (E(70) in several notable ways. For example, ATP hydrolysis by the activator is coupled to open complex formation with E(54, while no examples of such a requirement for ATP exist among the numerous genes transcribed by E(70. Towards this long term goal, the following shorter term objectives will be addressed in this research. First, DCTD mutants that are defective in activating transcription will be generated and characterized. For these experiments, a truncated form of DCTD (referred to as DCTDL143) that constitutively activates transcription and hydrolyzes ATP will be used. Mutant DCTDL143 proteins will be purified and analyzed for their abilities to activate transcription in vitro, hydrolyze ATP, bind DNA, and interact with E(54. DCTD can be crosslinked to (54 and the ( subunit of RNA polymerase, and interactions between DCTDL143 mutants and these subunits of E(54 will be examined using this crosslinking assay. These biochemical and genetic approaches will be valuable for dissecting protein-protein interactions required for transcriptional activation. Second, studies on the ATPase activity of DCTDL143 will be extended to include examination of potential inhibitors, determination of temperature and pH optima, and determination of dissociation constants for ATP. Photocrosslinking ATP (or analogs of ATP) to DCTD will be used to identify regions of the protein involved in ATP binding. Examining ATP binding and hydrolysis by DCTDL143 and DCTDLl43 mutants is likely to yield insights into how ATP hydrolysis is coupled to transcriptional activation. Together, these data will provide useful information on how DCTD activates transcription from the dctA promoter. %%% Despite the large number of transcriptional activators that have been identified in prokaryotic and eukaryotic organisms, the ways in which these proteins facilitate transcriptional activation are poorly understood. The long term goal of this laboratory is to characterize the molecular mechanism of transcriptional activation by a class of activators ((54 -dependent) in bacteria. One such activator, DCTD of Rhizobium meliloti will be studied. Mutant activators that are deficient in their ability to activate transcription will be generated using both random and localized mutagenesis. These mutant proteins will be examined for their abilities to activate transcription, bind and hydrolyze ATP, and interact with E(54. Studies on the ATPase activity of a truncated form of DCTD will be extended to include examination of potential inhibitors, determining temperature and pH optima for ATP hydrolysis, determining dissociation constants for MgATP, and identifying regions involved in MgATP binding. This work will help increase our understanding of how cells activate those genes which are appropriate for a given environment. ***