BIO-ORGANIC STUDIES ON SPECIFIC PROTEIN/DNA INTERFACES

特定蛋白质/DNA 界面的生物有机研究

• 批准号: 2459613
• 负责人: Alanna Schepartz
• 金额: $ 19.86万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459613
• 关键词: cAMP response element binding protein; chemical binding; chemical kinetics; conformation; gene expression; genetic enhancer element; intermolecular interaction; nucleic acid chemical synthesis; nucleic acid structure; protein structure; thermodynamics; transcription factor

Cellular phenomena such as replication, recombination, differentiation, and cell growth are regulated at athe most fundamental level by transcription factors, proteins that bind DNA and regulate gene expression. The direct relationship between aberrant gene expression and human disease emphasized the importance of understanding, at the molecular level, the mechanisms by which transcription factors discriminate between DNA sites. This proposal builds on discoveries made in our laboratory over the past year to analyze in detail the mechanisms by which eukaryotic bZIP (basic segment leucine zipper) transcription factors discriminate between the CRE and AP-1 sites, sequences that differ by the presence or absence of a single base pair. Despite their sequence similarity, the CRE and AP-1 sites are athe nuclear end-points of two different signal transduction pathways. We discovered that these DNA sites are equated structurally by an intrinsic bend in the CRE site, and that the CRE-selective bZIP protein CRE-BP1 overcomes this intrinsic bend and straightens the DNA using residues in its basic segment. We propose (Specific Aim 1) to identify which residues in the basic segment are required for distortion of the CRE site, and thereby explore the relationship between induced distortion and specificity. The observation that CRE-BP1 contains basic segment residues that stabilize a distorted form of the CRE site explains why these proteins bind the CRE site, but it does not explain why they prefer it. We will address this issue (Specific Aim 2) by engineering DNA minicircles to contain a CRE or P-1 site pre-bent towards the minor groove, into a conformation suitable for CRE-BP1. By comparing the affinity of CRE-BP1 for minicircles containing pre-bent DNA with the corresponding linear DNA fragments, we will learn whether specificity results from differential bending energies or differential DNA contact energies, or both. In addition, we propose to assess the generality of our "induced-straightening" model for the half-site spacing specificities of CREB/ATF proteins (Specific Aim 3) by examining other members of the family. We also propose a kinetic analysis of CRE/AP-1 discrimination by CRE-BP1 (Specific Aim 4). Finally, we propose an in vitro selection experiment (Specific Aim 5) to identify other DNA sequences that contain intrinsic major groove bends. Our long terms goals are to understand the thermodynamic basis for the specific protein.DNA and protein.protein interactions that orchestrate the precise control of gene expression. In a more specific sense, the relevance of these experiments to human medicine is straightforward: the three 21 base pair enhancer elements within the long terminal repeat of the human T-cell leukemia virus HTLV-1 each contain a CRE-like sequence, and the major T-cell proteins that bind the HTLVI21 bp repeats and mediate transactivation by the viral Tax transactivator are CREB/ATF family members. Therefore, results from the experiments described here will contribute directly to our thinking about the mechanisms of transcriptional activation by Tax.

细胞现象如复制、重组、分化和 细胞生长在最基本的水平上受转录调节 因子，结合DNA和调节基因表达的蛋白质。 直接 强调基因表达异常与人类疾病的关系 在分子水平上理解这些机制的重要性， 哪些转录因子能区分DNA位点。 这项建议 基于我们实验室过去一年的发现， 详细的机制，真核生物bZIP（基本段亮氨酸 拉链）转录因子区分CRE和AP-1位点， 存在或不存在单个碱基对的序列。 尽管它们的序列相似，但CRE和AP-1位点是同源核的。 两种不同信号转导途径的终点。 我们发现 这些DNA位点在结构上等同于DNA中的固有弯曲， CRE位点，并且CRE选择性bZIP蛋白CRE-BP1克服了这一点 固有的弯曲和拉直DNA使用其基本区段中的残基。 我们建议（具体目标1），以确定哪些残基在基本部分 ，从而探讨 诱导畸变和特异性之间的关系。 观察 CRE-BP1含有稳定扭曲的 CRE位点的形式解释了为什么这些蛋白质结合CRE位点，但它 没有解释为什么他们更喜欢它。我们将解决这个问题（具体 目的2）通过改造DNA微环，使其含有预弯曲的CRE或P-1位点， 朝向小沟，转变成适合CRE-BP1的构象。 通过 比较CRE-BP1对含有预弯曲DNA的微环的亲和力 与相应的线性DNA片段，我们将了解是否 特异性来自不同的弯曲能或不同的DNA 接触能量或两者。 此外，我们建议评估 我们的"诱导矫直"模型的一般性，为半场地间距 CREB/ATF蛋白的特异性（特异性目标3），通过检查其他 家庭成员。 我们还提出了CRE/AP-1的动力学分析 CRE-BP1（具体目标4）的区分。 最后，我们提出了一个在 体外选择实验（特定目标5），以鉴定其他DNA序列 包含内在的主沟弯曲。 我们的长期目标是 了解特定蛋白质的热力学基础。DNA和 蛋白质相互作用协调了基因的精确控制 表情 从更具体的意义上说，这些实验的相关性 对人类医学来说很简单：三个21个碱基对的增强子 人类T细胞白血病病毒长末端重复序列内的元件 HTLV-1每一个都含有一个CRE样序列，并且主要的T细胞蛋白， 结合HTLVI21 bp重复序列并介导病毒Tax的反式激活 反式激活因子是CREB/ATF家族成员。 因此，结果从 这里描述的实验将直接有助于我们思考 Tax的转录激活机制。