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BIO-ORGANIC STUDIES ON SPECIFIC PROTEIN/DNA INTERFACES

特定蛋白质/DNA 界面的生物有机研究

基本信息

批准号：
2750009
负责人：
Alanna Schepartz
金额：
$ 20.33万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/2750009
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

项目摘要

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，例如复制、重组、分化和细胞生长在最基本的水平上通过转录进行调节因子，即结合 DNA 并调节基因表达的蛋白质。直接的强调异常基因表达与人类疾病之间的关系在分子水平上理解其机制的重要性哪些转录因子可以区分 DNA 位点。这个提议基于我们实验室过去一年的发现来分析详细介绍了真核生物 bZIP（基本片段亮氨酸拉链）转录因子区分 CRE 和 AP-1 位点，因存在或不存在单个碱基对而不同的序列。尽管 CRE 和 AP-1 位点序列相似，但它们是非核位点。两个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中的内在弯曲 CRE 位点，而 CRE 选择性 bZIP 蛋白 CRE-BP1 克服了这一点使用其基本片段中的残基来内在弯曲和拉直 DNA。我们建议（具体目标 1）识别基本片段中的哪些残基需要扭曲CRE位点，从而探索诱导畸变和特异性之间的关系。观察结果 CRE-BP1 含有稳定扭曲的基本片段残基 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（具体目标 2) 通过改造 DNA 小环以包含预弯曲的 CRE 或 P-1 位点朝向小沟，形成适合 CRE-BP1 的构象。经过比较 CRE-BP1 对含有预弯曲 DNA 的小环的亲和力与相应的线性 DNA 片段，我们将了解是否差异弯曲能或差异 DNA 产生特异性接触能量，或两者兼而有之。此外，我们建议评估我们的半位点间距“诱导矫直”模型的一般性通过检查其他蛋白来确定 CREB/ATF 蛋白的特异性（具体目标 3）家庭成员。我们还提出了 CRE/AP-1 的动力学分析 CRE-BP1 的歧视（具体目标 4）。最后，我们提出一个体外选择实验（具体目标 5）鉴定其他 DNA 序列包含固有的主要凹槽弯曲。我们的长期目标是了解特定蛋白质的热力学基础。DNA 和蛋白质与蛋白质之间的相互作用，协调基因的精确控制表达。从更具体的意义上讲，这些实验的相关性对人类医学来说很简单：三个21碱基对增强子人类 T 细胞白血病病毒长末端重复序列中的元件 HTLV-1 各自包含 CRE 样序列，以及主要的 T 细胞蛋白结合 HTLVI21 bp 重复序列并通过病毒 Tax 介导反式激活反式激活因子是 CREB/ATF 家族成员。因此，结果由这里描述的实验将直接有助于我们思考 Tax 转录激活机制。