Transcriptional Activation with Sigma54-Holoenzyme

摘要MCB 9974558 PI：Timothy Hoover苜蓿中华根瘤菌C4-二羧酸蛋白D（DctD）将用作研究sigma 54-RNA聚合酶全酶转录激活的模型。 DctD与位于dctA启动子上游的增强子位点结合，然后通过DNA成环与结合在启动子处的σ 54全酶接触以激活转录。 在高浓度下，DctD可以在没有增强子的情况下通过直接从溶液中接触σ 54全酶来激活转录。 DctD的一些突变形式在缺乏增强子的情况下不能激活转录，并且这些突变蛋白质可能具有降低的对σ 54全酶的亲和力。 在第一个具体目标中，增强子依赖性DctD突变体和σ 54全酶之间的相互作用将通过等温滴定量热法和表面等离子体共振来检查，以确定增强子依赖性DctD突变体是否对σ 54全酶具有降低的亲和力。 此外，还将研究抑制增强子依赖性DctD突变体的sigma 54全酶的突变形式。 从这些研究中获得的信息将有助于确定参与蛋白质-蛋白质相互作用的位点。 在第二个具体目标中，将分离并在体外表征在σ 54全酶与启动子结合后的步骤中有缺陷的σ 54的突变形式。 这些研究将有助于阐明sigma 54在转录激活中的作用。 最后，幽门螺杆菌FlgR（鞭毛调节蛋白）将在体外纯化和表征。 FlgR是σ 54全酶的激活剂，但它缺乏在其他激活剂中发现的DNA结合结构域。 FlgR可能在生理浓度下直接从溶液中接触σ 54全酶以催化开放复合物的形成，或者它含有负责DNA结合的另一个亚基。 许多细菌含有sigma 54-RNA聚合酶全酶，这种形式的全酶负责基因的表达，这些基因的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，和菌毛蛋白形成。 这些微生物过程中的许多具有显著的环境、工业、农业或医学影响。 了解sigma 54依赖基因是如何调节的，对于开发和控制负责这些微生物活动的细菌至关重要。

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.