Transcriptional Activation with Sigma54-Holoenzyme

使用 Sigma54-Holoenzyme 进行转录激活

• 批准号: 9974558
• 负责人: Timothy Hoover
• 金额: $ 32万
• 依托单位: University of Georgia Research Foundation Inc
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9974558&HistoricalAwards=false
• 关键词: Transcriptional; Activation; Sigma54; Holoenzyme

ABSTRACTMCB 9974558PI: Timothy HooverSinorhizobium meliloti C4-dicarboxylic acid protein D (DctD) will be used as a model to study transcriptional activation with sigma54-RNA polymerase holoenzyme. DctD binds to enhancer sites located upstream of the dctA promoter and then contacts sigma54-holoenzyme bound at the promoter through DNA looping to activate transcription. At high concentrations, DctD can activate transcription in the absence of the enhancer by contacting sigma54-holoenzyme directly from solution. Some mutant forms of DctD cannot activate transcription in the absence of the enhancer, and these mutant proteins may have reduced affinities for sigma54-holoenzyme. In the first specific aim, interactions between enhancer-dependent DctD mutants and sigma54-holoenzyme will be examined by isothermal titration calorimetry and surface plasmon resonance to determine if the enhancer-dependent DctD mutants have reduced affinities for sigma54-holoenzyme. In addition, a search will be made for mutant forms of sigma54-holoenzyme that suppress enhancer-dependent DctD mutants. Information gained from these studies will help identify sites that are involved in protein-protein interactions. In the second specific aim, mutant forms of sigma54 that are defective in steps following binding of sigma54-holoenzyme to the promoter will be isolated and characterized in vitro. These studies will help elucidate the role of sigma54 in transcriptional activation. Finally, Helicobacter pylori FlgR (flagellar regulatory protein) will be purified and characterized in vitro. FlgR is an activator of sigma54-holoenzyme but it lacks the DNA-binding domain found in other activators. FlgR presumably either contacts sigma54-holoenzyme directly from solution at physiological concentrations to catalyze open complex formation, or it contains another subunit that is responsible for DNA binding. Both possibilities are unusual for activators of sigma54-holoenzyme and experiments will be done to distinguish between the two.Many bacteria contain sigma54-RNA polymerase holoenzyme, and this form of holoenzyme is responsible for the expression of genes whose products are involved in a variety of important microbial processes, including nitrogen assimilation, nitrogen fixation, toluene degradation, hydrogen metabolism, flagellar biosynthesis, and pilin formation. Many of these microbial processes have significant environmental, industrial, agricultural or medical implications. Understanding how sigma54-dependent genes are regulated is critical for exploiting and controlling the bacteria that are responsible for these microbial activities.

本文以Timothy HooverSinorhizobium Meliloti C4-二羧酸蛋白D(DctD)为模型，研究了Sigma54-RNA聚合酶全酶对转录的激活作用。DctD与位于DCTA启动子上游的增强子结合，然后通过DNA环与结合在启动子上的sigma54-全酶接触来激活转录。在高浓度时，DctD可以在没有增强子的情况下，通过直接从溶液中接触sigma54-全酶来激活转录。DctD的某些突变形式在没有增强子的情况下不能激活转录，这些突变蛋白可能降低了对Sigma54-全酶的亲和力。在第一个特定目标中，将通过等温滴定量热法和表面等离子体共振来检测依赖于增强子的DctD突变体与sigma54-全酶之间的相互作用，以确定依赖于增强子的DctD突变体是否降低了对sigma54-全酶的亲和力。此外，还将搜索抑制增强子依赖的DctD突变体的Sigma54-全酶的突变形式。从这些研究中获得的信息将有助于确定与蛋白质相互作用有关的位点。在第二个特定目标中，将在体外分离和鉴定在sigma54-全酶与启动子结合后存在步骤缺陷的sigma54突变形式。这些研究将有助于阐明sigma54在转录激活中的作用。最后，幽门螺杆菌FlgR(鞭毛调节蛋白)将在体外得到纯化和鉴定。FlgR是Sigma54-全酶的激活剂，但它缺乏其他激活剂中的DNA结合结构域。FlgR可能要么直接从生理浓度的溶液中接触sigma54-全酶，以催化开放的复合体形成，要么它含有另一个负责DNA结合的亚基。对于sigma54-全酶的激活剂来说，这两种可能性都是不寻常的，将进行实验来区分这两种情况。许多细菌都含有sigma54-RNA聚合酶全酶，这种形式的全酶负责基因的表达，其产物参与各种重要的微生物过程，包括氮同化、固氮、甲苯降解、氢代谢、鞭毛生物合成和菌毛形成。其中许多微生物过程对环境、工业、农业或医学都有重大影响。了解依赖sigma54的基因是如何调控的，对于开发和控制负责这些微生物活动的细菌至关重要。