BIO-ORGANIC STUDIES ON SPECIFIC PROTEIN/DNA INTERFACES

细胞现象，如复制、重组、分化和 细胞的生长由转录在最基本的水平上进行调节。 因子，结合DNA并调节基因表达的蛋白质。直接式 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 哪些转录因子区分DNA位点。这项建议 以我们实验室过去一年的发现为基础进行分析 详细介绍了真核生物bZIP(碱性片段亮氨酸)的作用机制 Zipper)转录因子区分Cre和AP-1位点， 因单个碱基对的存在或缺失而不同的序列。 尽管它们的序列相似，但Cre和AP-1位点是核的 两条不同信号转导通路的终点。我们发现 这些DNA位点在结构上等同于 Cre结合位点，并且Cre选择性bZIP蛋白Cre-BP1克服了这一点 利用DNA基本片段中的残基，固有地弯曲和拉直DNA。 我们建议(具体目标1)确定基本片段中的哪些残基 是造成CRE站点扭曲所必需的，从而探索 诱发失真与特异性的关系。观察到的 Cre-BP1含有碱性片段残基，可以稳定扭曲的 Cre位点的形式解释了为什么这些蛋白质结合Cre位点，但它 这并不能解释为什么他们更喜欢它。我们将解决此问题(具体 目的2)通过改造DNA微环来包含Cre或P-1位点预弯 朝向次要凹槽，形成适合CRE-BP1的构象。通过 Cre-BP1对含有预弯DNA微环的亲和力比较 通过相应的线状DNA片段，我们将了解到 特异性来自不同的弯曲能或不同的DNA 接触能量或两者兼而有之。此外，我们建议评估 我们的半场地间距“诱导矫直”模型的一般性 CREB/ATF蛋白的特异性(特异靶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，我们提出了一个新的 用于鉴定其他DNA序列的体外选择实验(特定目标5) 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--协调精确控制基因的蛋白质相互作用 表情。在更具体的意义上，这些实验的相关性 对人类医学来说是直截了当的：三个21碱基对增强剂 人T细胞白血病病毒长末端重复序列中的元件 HTLV-1每个病毒都包含一个类似Cre的序列，并且主要的T细胞蛋白 结合HTLVI21BP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，来自 这里描述的实验将直接有助于我们思考 税收激活转录的机制。

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.