Molecular Origins of Specificity in Protein-Nucleic Acid Interactions

蛋白质-核酸相互作用特异性的分子起源

• 批准号: 9816578
• 负责人: Jannette Carey
• 金额: $ 36.01万
• 依托单位: Princeton University
• 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: 1999-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9816578&HistoricalAwards=false
• 关键词: Molecular; Origins; Specificity; Protein; Nucleic

Carey 98-16578Thr arginine repressor of E. coli, ArgR, is the transcriptional transducer of intracellular L-arginine concentration, and the master regulator of a large group of genes involved in arginine biosynthesis, the arginine regulon. ArgR is unusual among repressors because it binds DNA as a 98 kDa hexamer and functions as both a repressor and an accessory factor in resolution of plasmid ColE1 multimers by intramolecular recombination. Arginine binding increases the affinity and specificity of DNA binding by an unknown mechanism. ArgR is organized into a C-terminal domain (ArgRC) that houses the hexamerization and arginine-binding functions, and an N-terminal domain (ArgRN) which in isolation retains DNA-binding ability but is monomeric and arginine-independent. The structures of the two domains are known separately, and on the basis of these structures certain mechanisms of allosteric activation used by other regulatory proteins can be ruled out for ArgR. The separate structures of the domains, combined with biochemical and biophysical data obtained during the previous funding period on DNA binding by intact ArgR, were used to derive a model for the hexameric ArgR-DNA complex. This model and other recent progress clearly implicates subunit assembly, interdomain interactions, and conformational changes transmitted through the interdomain linker as key elements of the allosteric activation mechanism, and suggests that interactions between DNA-binding domains may confer cooperativity on the binding reaction. These hypotheses will be examined in this study with four specific aims. 1. Stoichiometry and cooperativity of DNA binding. The current model implicates only four of the six equivalent subunits of ArgR in binding to typical operator sites, which contain four half-sites arranged as tandem palindromes. The model thus implies that ArgR subunits may cooperate at pairwise or higher levels, and that a third pair of ArgR subunits is available to take up a third DNA palindrome. These features will be examined using mainly quantitative gel retardation with a set of short synthetic DNAs and cloned constructs containing various half-site arrangements to determine the stoichiometry and cooperativity of various binding modes for ArgR. The orientation of subunits on the DNA is crucial to their interactions, and will be examined following site-directed mutagenesis to permit disulfide bonding between two monomers. 2. Subunit assembly equilibria and conformational changes. Subunit assembly will be examined for the wildtype protein in the presence and absence of L-arginine by using sedimentation equilibrium analysis in the ultracentrifuge. In addition, a combination of sedimentation velocity and sedimentation equilibrium analysis will be used to probe possible global conformational changes upon L-arginine binding. 3. Role of interdomain interactions and linker in allosteric activation. Six superrepressor mutants were isolated that lie in the N-terminal domain or linker, at or near the interface with the C-terminal domain. The superrepressor phenotype suggests that transmission of the L-arg binding signal from the C-terminal domain to the DNA-binding domain is affected by the mutations. Each of these mutants will be cloned, overexpressed, and purified, and characterized by a battery of quantitative biochemical and biophysical tools to assess their effects on allosteric activation by studying their DNA- and L-arginine-binding and multimerization equilibria. 4. Global regulatory function. Functional similarities to other systems suggest that ArgR may be a previously unrecognized global regulator and/or an activator. Database searching identified several putative operator sites in genes not previously recognized as part of the arg regulon, and selective DNA binding to three of these has been confirmed. The effects of ArgR on gene expression in these three systems will be examined using b-gal fusions in various ArgR backgrounds, and the details of DNA binding will be examined in vitro.2. Non-technical Gene expression is controlled by regulatory proteins that recognize specific DNA sequences. The mechanisms responsible for sequence-specific recognition are of fundamental interest to extend our understanding of molecular function, and also of potential practical interest for the prospect of influencing recognition events, for example in cases where expression of disease genes might be controlled. Bacterial systems provide a good model for understanding specificity because metabolically related genes are often coordinately controlled by one master regulatory protein that must recognize a group of similar but not identical DNA sequences, while maintaining the ability to reject slightly more distantly related sequences. Such systems often achieve enhanced specificity through interactions with small ligands that act as co-effectors. The molecular mechanisms through which co-effectors confer specificity are quite varied, and many apparently unique examples are known. In E. coli, the metabolism of arginine, a required amino acid, is controlled by the arginine repressor, ArgR. Arginine itself serves as a co-effector: it binds to ArgR and increases the specificity of DNA recognition by an unknown mechanism. Previous work by P.I. identified structural units within ArgR that house the various functions of the protein, and indicated that of six equivalent units, four cooperate in arginine-specific DNA recognition. This cooperation requires communication among the protein's structural units. In this study, the mechanisms of this communication will be examined. The communication will first be described in quantitative terms by studying the DNA recognition reaction directly, using methods developed previously. Interactions between units will be characterized by determining the size and shape of the molecule in solution under various conditions. A crucial interfacial region of the protein has also been identified and will be probed by studying DNA recognition using proteins that are mutated in this region. This work is expected to extend our understanding of the mechanisms leading to selective DNA binding.

大肠杆菌的thr精氨酸抑制因子（ArgR）是细胞内l -精氨酸浓度的转录转换器，也是一大批参与精氨酸生物合成的基因（精氨酸调节因子）的主要调控因子。ArgR在抑制因子中是不寻常的，因为它以98 kDa的六聚体结合DNA，并且在通过分子内重组拆分质粒ColE1多聚体时既作为抑制因子又作为辅助因子。精氨酸结合以一种未知的机制增加了DNA结合的亲和力和特异性。ArgR由c端结构域（ArgRC）和n端结构域（ArgRN）组成，前者具有六聚化和精氨酸结合功能，后者保留dna结合能力，但不依赖于单体和精氨酸。这两个结构域的结构是分别已知的，基于这些结构，可以排除ArgR的其他调节蛋白使用的某些变构激活机制。这些结构域的独立结构，结合在之前资助期间获得的完整ArgR DNA结合的生化和生物物理数据，用于推导六聚体ArgR-DNA复合物的模型。该模型和其他最新进展清楚地表明，亚基组装、结构域间相互作用和通过结构域间连接体传递的构象变化是变构激活机制的关键要素，并表明dna结合结构域之间的相互作用可能赋予结合反应的协同性。这些假设将在本研究中以四个具体目标进行检验。1. DNA结合的化学计量学和协同性。目前的模型表明，ArgR的6个等效亚基中只有4个与典型的操作位点结合，这些操作位点包含4个排列成串联回文的半位点。因此，该模型表明ArgR亚基可能在成对或更高水平上合作，并且第三对ArgR亚基可用于占用第三个DNA回文。这些特征将主要通过一组短合成dna和含有各种半位点排列的克隆构建物的定量凝胶阻滞来检验，以确定ArgR的各种结合模式的化学计量学和协同性。DNA上亚基的方向对它们的相互作用至关重要，并将在位点定向诱变后进行检查，以允许两个单体之间的二硫键结合。2. 亚基组装平衡和构象变化。利用超离心沉降平衡分析，在存在和不存在l -精氨酸的情况下，检测野生型蛋白的亚基组装。此外，将结合沉降速度和沉降平衡分析来探讨l -精氨酸结合后可能发生的全局构象变化。3. 结构域间相互作用和连接物在变构活化中的作用。6个超抑制突变体位于n端结构域或连接体中，在与c端结构域的界面处或附近。超抑制因子表型表明，L-arg结合信号从c端结构域传递到dna结合结构域受到突变的影响。每个突变体都将被克隆、过表达和纯化，并通过一系列定量生化和生物物理工具进行表征，通过研究它们的DNA和l -精氨酸结合和多化平衡来评估它们对变构激活的影响。4. 全球监管功能。与其他系统的功能相似性表明，ArgR可能是一种以前未被识别的全局调节剂和/或激活剂。数据库检索发现了几个先前未被认为是arg调控子一部分的基因中的假定操作位点，并且已确认其中三个位点的选择性DNA结合。ArgR对这三种系统中基因表达的影响将在不同ArgR背景下使用b-gal融合来研究，DNA结合的细节将在体外进行研究。非技术基因表达是由识别特定DNA序列的调节蛋白控制的。序列特异性识别的机制对于扩展我们对分子功能的理解具有重要意义，并且对于影响识别事件的前景具有潜在的实际意义，例如在疾病基因表达可能被控制的情况下。细菌系统为理解特异性提供了一个很好的模型，因为代谢相关基因通常由一个主调控蛋白协调控制，该蛋白必须识别一组相似但不相同的DNA序列，同时保持拒绝稍远相关序列的能力。这类系统通常通过与充当协同效应的小配体相互作用来增强特异性。共同效应物赋予特异性的分子机制各不相同，并且已知许多明显独特的例子。在大肠杆菌中，精氨酸（一种必需的氨基酸）的代谢由精氨酸抑制因子ArgR控制。精氨酸本身作为一种协同效应：它与ArgR结合，并通过一种未知的机制增加DNA识别的特异性。P.I.先前的工作鉴定了ArgR中的结构单元，这些结构单元容纳了蛋白质的各种功能，并指出在6个等效单元中，有4个在精氨酸特异性DNA识别中合作。这种合作需要蛋白质结构单元之间的交流。在这项研究中，这种交流的机制将被检查。通过使用先前开发的方法直接研究DNA识别反应，将首先以定量术语描述这种通信。单元之间的相互作用将通过确定不同条件下溶液中分子的大小和形状来表征。该蛋白的一个关键界面区域也已被确定，并将通过使用该区域突变的蛋白质研究DNA识别来进行探测。这项工作有望扩展我们对选择性DNA结合机制的理解。