Biochemical Analysis of PHO5 Gene Regulation

本研究将探讨核心组蛋白在RNA聚合酶II转录调控中的功能。许多，如果不是大多数，转录调控机制可能只在染色质背景下完全表现出来。核小体形式的核心组蛋白抑制转录。最近的遗传学研究表明，核心组蛋白直接参与RNA聚合酶II的转录调控。以Pho4p转录因子激活酵母PHOS启动子为模型，研究染色质模板上的转录调控机制。这些研究的结果将对核心组蛋白在决定基因是活跃还是被抑制的机制中的作用提供更精确的理解。体内核心组蛋白的缺失导致酵母PHO5、GAL 1和CYC 1基因的转录降低。以往对PHO5基因调控的研究为研究转录调控染色质的机制提供了重要线索。然而，这些研究都是在体内进行的。要更全面地了解这一机制，就需要进行生化分析。该实验室是第一个纯化酵母核心组蛋白的实验室，其数量足以用于对其转录调节作用的生化研究。这些组蛋白被用来重建核小体模板，恢复PHO5启动子基础转录的抑制。因此，这个实验室在进行这些分析方面处于独特的地位。本研究将：(1)重建PHO5启动子的转录激活。体外转录将由重组和纯化的酵母基础转录因子以及纯化的Pho4p和Pho2p组成，这些系统将用于重建基础和激活的PHO5转录。(2)通过重组核小体确定PHO5基础转录抑制程度。核小体将使用纯化的组分在PHOS启动子上重组。将比较裸DNA和染色质模板之间的基础转录水平，并确定染色质模板上TATA-box和RNA起始位点可及性与转录活性之间的关系。(3)研究Pho4p对PHO5启动子的核小体抑制的抑制能力。核小体将被重组到PHO5启动子上，Pho4p抵消核小体抑制所需的因子将被确定。在裸模板和染色质模板上比较PHO5的激活水平。本研究探讨了ATP合成酶的结构、功能和调控之间的关系。这种变构酶可逆地利用跨膜质子梯度的能量合成ATP。该研究的长期目标是有助于理解能量转导过程的机制，并了解该酶在植物和动物的生理条件下是如何调节的。NSF先前的资助已经导致：1)克隆和过表达叶绿体ATP合成酶（CF1）催化F1部分的所有五个亚基；2)从细菌包涵体中回收亚基并将其重新折叠成其原生的活性形式；3)重组成功能性F1复合物。进一步的研究将利用该系统对CF1亚基进行基因工程，以分析功能重要区域，并使用生化和生物物理技术检查亚基在催化过程中的功能动力学。本提案描述了一种多学科协作方法，重点关注三个较小的CF1亚基（）和（）的结构和功能。基因工程实验将包括：1)位点定向诱变研究，通过替换或删除特定氨基酸或氨基酸序列来探索多肽链不同区域的功能和结构重要性；2)通过将内源性（色氨酸）和外源性（通过半胱氨酸残基）荧光探针附着在（和）亚基上的特定位点来研究亚基动力学。标记的亚基将与这些亚基中的CF1缺陷进行重组，用于荧光研究，旨在(a)使用荧光共振能量转移测量CF1复合物内亚基的结构映射，以及(b)通过监测内源和外源荧光探针的时间相关各向异性来监测酶催化转换过程中的亚基动力学。3)化学交联研究，涉及双功能化学交联剂在（和）亚基内的各种工程位点的附着。突变亚基将与其他CF1亚基以及酶的膜结合质子通道部分（CF0）重建，用于化学交联研究，旨在确定CF1和CF0亚基之间的相互作用位点。本研究探讨了植物叶绿体膜ATP合酶的化学结构与生物学功能之间的关系。这种酶负责将来自阳光的能量转化为化学储存形式。ATP（三磷酸腺苷）反过来，ATP为植物将二氧化碳转化为糖提供能量。在动物和细菌中，类似的酶提供超过90%的能量依赖性代谢过程所需的ATP。本提案中描述的研究的长期目标是在分子水平上确定ATP合酶如何捕获能量并将其转化为ATP。我们打算通过基因工程ATP合酶来实现这一目标，以探索酶内特定位点对能量转换（催化）过程的重要性。一些基因工程位点将为化学探针的附着提供位点，这些探针将用于识别在催化过程中发生的涉及酶结构变化的特定事件。将其他化学探针附着在基因工程位点上，将使我们能够将酶的片段交联在一起，这样我们就可以确定酶内蛋白质-蛋白质相互作用的特定位点，这些位点对催化作用很重要。彻底了解这种非常有效的自然节能过程将有可能导致设计和实施大大改进的能量储存和利用过程或人类使用。

This research will investigate the function of the core histones in the regulation of RNA polymerase II transcription. Many, if not most, transcription regulatory mechanisms may be fully manifest only in a chromatin context. Core histones in the form of nucleosomes repress transcription. Recent genetics studies indicate that core histones are directly involved in RNA polymerase II transcription regulation. The mechanism of transcription regulation on a chromatin template will be investigated using Pho4p transcription factor activation of the yeast PHOS promoter as a model. Results from these studies will provide a more precise understanding of the role of the core histones in the mechanisms that determine whether a gene is active or repressed. Depletion of core histones in vivo causes the derepression of transcription of the yeast PHO5, GAL 1, and CYC 1 genes. Previous studies of PHO5 gene regulation have provided important clues to the mechanism of transcription regulation on chromatin. However, these studies have all been carried out in vivo. A more complete understanding o f the mechanism requires biochemical analysis. This laboratory is the first to have purified yeast core histones in large enough quantities for biochemical investigation of their role transcription regulation. These histones have been used to reconstitute nucleosomal templates, restoring repression of basal transcription to the PHO5 promoter. Thus, this laboratory is in a unique position to undertake these analyses. This research will: (1) Reconstitute transcription activation from the PHO5 promoter. In vitro transcription will be reconstituted from recombinant and purified yeast basal transcription factors and purified Pho4p and Pho2p, and these systems will be used to reconstitute basal and activated PHO5 transcription. (2) Determine the degree of PHO5 basal transcription repression by reconstituted nucleosomes. Nucleosomes will be reconstituted on the PHOS promoter using purified components. The levels of basal transcription between naked DNA and chromatin templates will be compared, and the relation between TATA-box and RNA start site accessibility and transcription activity on chromatin templates will be determined. (3) Investigate the ability of Pho4p to counteract nucleosome repression of the PHO5 promoter. Nucleosomes will be reconstituted onto the PHO5 promoter, and the factor requirement will be determined for Pho4p to counteract nucleosome repression. The level of PHO5 activation will be compared on naked and chromatin templates. * * * 9506255 Richter This research addresses the relationship between the structure, function and regulation of the ATP synthase enzyme. This allosteric enzyme reversibly utilizes the energy of a transmembrane proton gradient to synthesize ATP. The long term goal of the research is to contribute toward an understanding of the mechanism of the energy transduction process and to understand how this enzyme is regulated under physiological conditions in plants and animals. Prior NSF funding has led to: 1) Cloning and over-expression of all five subunits of the catalytic F1 portion of the chloroplast ATP synthase (CF1); 2) Recovery of the subunits from bacterial inclusion bodies and refolding them into their native, active forms; and 3) Reconstituting them into a functional F1 complex. Further studies will utilize this system to genetically engineer CF1 subunits for analyzing regions of functional importance, and to examine functional dynamics of subunits during the catalytic process using biochemical and biophysical techniques. This proposal describes a collaborative multidiciplinary approach focusing on the structure and function of the three smaller CF1 subunits, )and (. Genetic engineering experiments will involve: 1) Site-directed mutagenesis studies to probe the functional and structural importance of different regions of the polypeptide chains via replacement or deletion of specific amino acids or sequences of amino acids; 2) Studies of subunit dynamics through attachment of intrinsic (tryptophans) and extrinsic (via cysteine residues) fluorescent probes to specific sites on the ( ( and ( subunits. Labeled subunits will be reconstituted with CF1 deficient in these subunits for fluorescence studies aimed at (a) structural mapping of subunits within the CF1 complex using fluorescence resonance energy transfer measurements and (b) monitoring subunit dynamics during catalytic turnover by the enzyme by monitoring the time- dependent anisotropy of intrinsic and extrinsic fluorescent probes; 3) Chemical crosslinking studies involving attachment of bifunctional chemical crosslinking agents at various engineered sites within the ( and ( subunits. Mutant subunits will be reconstituted with the other CF1 subunits and with the membrane-bound proton channel portion (CF0) of the enzyme for chemical crosslinking studies aimed at identifying the sites of interaction between CF1 and CF0 subunits. %%% This research addresses the relationship between the chemical structure and biological function of the ATP synthase enzyme of plant chloroplast membranes. This enzyme is responsible for the conversion of the energy from sunlight into the chemical storage form. ATP (adenosine triphosphate). ATP in turn supplies the energy for the conversion of carbon dioxide into sugar in plants. Analogous enzymes supply more than 90% of the ATP required for energy-dependent metabolic processes in animals and bacteria. The long term goal of the research described in this proposal is to identify, at the molecular level, how the ATP synthase enzymes capture energy and convert it into ATP. We intend to approach this goal by genetically engineering the ATP synthase so as to probe the importance of specific sites within the enzyme for the energy conversion (catalytic) process. Some of the genetically engineered sites will provide sites for attachment of chemical probes which will be used to identify specific events involving changes in the structure of the enzyme which occur during the catalytic process. Attachment of other chemical probes to genetically engineered sites will allow us to cross-link (tether) pieces of the enzyme together so that we can identify specific sites of protein-protein interaction which occur within the enzyme and which are important for catalysis. A thorough understanding of this very efficient natural energy conserving process will potentially lead to the design and implementation of vastly improved energy storage and utilization processes or human use.