Transcriptional Activation by Rhizobium meliloti DCTD
苜蓿根瘤菌 DCTD 的转录激活
基本信息
- 批准号:9506333
- 负责人:
- 金额:$ 9.21万
- 依托单位:
- 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:1995-09-15 至 1997-08-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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. ***
9506333 Hoover苜蓿根瘤菌DCTD激活来自dctA(其编码C4-二羧酸转运蛋白)的转录,并且属于β 4-依赖性激活因子家族,其可能通过共同机制起作用。 Sigma 54依赖性基因编码参与重要和多样的代谢过程的产物,包括氮同化、固氮、C4-二羧酸转运、甲苯降解、氢代谢和菌毛蛋白形成。 本实验室的长期目标是了解转录激活的分子机制(54 - 依赖性激活剂。 研究β 5 4依赖性激活因子如DCTD如何促进转录激活将极大地有助于理解细菌基因调控和生理学。 β 4-RNA聚合酶全酶(E(54))的转录机制在几个值得注意的方面与主要形式的RNA聚合酶全酶(E(70))的转录机制显著不同。 例如,激活剂的ATP水解与E(54)的开放复合物形成偶联,而在由E(70)转录的众多基因中不存在这种对ATP的需求的实例。 为了实现这一长期目标,本研究将解决以下短期目标。 首先,将产生并表征在激活转录方面有缺陷的DCTD突变体。 对于这些实验,将使用组成型激活转录并水解ATP的截短形式的DCTD(称为DCTDL 143)。 将纯化突变体DCTDL 143蛋白,并分析其体外激活转录、水解ATP、结合DNA和与E相互作用的能力(54. DCTD可以与β 4和RNA聚合酶的β亚基交联,并且DCTDL 143突变体与β 4的这些亚基之间的相互作用将使用该交联测定来检查。 这些生物化学和遗传学的方法将是有价值的解剖蛋白质-蛋白质相互作用所需的转录激活。 其次,对DCTDL 143的ATP酶活性的研究将扩展到包括检查潜在的抑制剂,确定最佳温度和pH值,并确定ATP的解离常数。 将ATP(或ATP类似物)光交联至DCTD将用于鉴定参与ATP结合的蛋白质的区域。 检查DCTDL 143和DCTDL 143突变体的ATP结合和水解可能产生对ATP水解如何与转录激活偶联的见解。 总之,这些数据将提供有关DCTD如何激活dctA启动子转录的有用信息。 尽管在原核生物和真核生物中已经鉴定出大量的转录激活因子,但对这些蛋白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因子(54 -依赖性)转录激活的分子机制。 苜蓿根瘤菌的DCTD是一种活性物质。 将使用随机和局部诱变两者来产生在其激活转录的能力方面有缺陷的突变激活剂。 将检查这些突变蛋白质激活转录、结合和水解ATP以及与E相互作用的能力(54. 对截短形式的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。 ***
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Timothy Hoover其他文献
Timothy Hoover的其他文献
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{{ truncateString('Timothy Hoover', 18)}}的其他基金
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Genome-Wide Analysis of the Salmonella RpoN Regulon
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1051175 - 财政年份:2011
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REU Site: Research in Prokaryotic Biology
REU 网站:原核生物学研究
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0453353 - 财政年份:2005
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Standard Grant
Microbial Genome Sequencing: Genome Sequencing of the Budding Bacterium Hyphomonas Neptunium
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0237224 - 财政年份:2002
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Transcriptional Activation with Sigma54-Holoenzyme
使用 Sigma54-Holoenzyme 进行转录激活
- 批准号:
9974558 - 财政年份:1999
- 资助金额:
$ 9.21万 - 项目类别:
Continuing Grant
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- 批准号:
9630454 - 财政年份:1996
- 资助金额:
$ 9.21万 - 项目类别:
Continuing Grant
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