STRUCTURAL CHARACTERIZATION OF SP1-DNA COMPLEXES

RNA聚合酶II合成信使RNA需要 大量的辅助转录因子， 结合到位于真核细胞上游的特异性启动子DNA序列上 基因. 这些转录因子调节启动 通过组合和接合以时间顺序的方式转录 活性转录复合物 为了了解详细的 由于转录因子的作用，已经做出了努力， 这些因子是重建转录所必需的 体外活性。 这些实验导致了 鉴定、纯化和表征一种这样 启动子特异性转录因子Sp1。 Sp l 增强多种病毒和细胞基因的转录， 结合到一个或多个“GC盒”识别元件（包含 六核核心GGGCGG）在5'侧翼启动子序列内 通过使用三个“锌指”域。 一般来说，DNA结合表面被设计成具有高度限定的分子量。 对于它们的同源DNA结合位点的偏好; Spl是其中独特的。 迄今为止发现的转录因子，因为它识别许多 转录激活结合位点，其可以被分类为 高、中或低亲和力。 因此，我们对以下内容非常感兴趣 检查Sp1与“GC盒”序列的结合， 这些因素导致了这一异常混杂的序列 识别能力 SPL系统可以进行详细的检查 由于其DNA结合特性定位于“锌指”， 域 该提案描述了一种方法，使我们能够 利用相对紧凑的“锌指”基序来定义那些 结构因素负责识别多样性的Sp 1。 我们的目标是：（1）表达可溶性Sp1短片段， 含有三个“锌指”结构域，（2）为了测试这些结构域的能力， 片段复制完整Spl的天然结合特性， 带移分析，（3）表达和纯化大量的那些 显示Spl结合能力的肽，（4）定量地 表征这些片段与双链体的相互作用 寡核苷酸的分子生物学（带移测定，竞争 分析，诱变研究）和生物物理技术（量热法， NMR），以确定所观察到的化合物的化学/结构基础。 DNA结合的多样性，以及（5）确定溶液结构 通过NMR光谱技术分析活性Spl片段。 我们的长期目标是表征溶液结构， 使用一系列高和中等亲和力的Spl-DNA复合物 DNA识别序列。 这些数据不仅有助于我们 在分子水平上理解“锌指”结构域是如何 以及这个主要的结构基序是如何 能够识别高度可变的DNA序列。

Synthesis of messenger RNA by RNA polymerase II requires the interaction of a large array of auxiliary transcription factors that recognize and bind to specific promoter DNA sequences located upstream of eukaryotic genes. These transcription factors regulate the initiation of transcription in a temporally ordered manner by assembling and engaging the active transcription complex. In order to understand the detailed roles played by transcription factors, efforts have been made to fractionate the factors necessary to reconstitute transcriptional activity in vitro. These experiments have resulted in the identification, purification and characterization of one such promoter-specific transcription factor, Sp l, from HeLa cells. Sp l enhances transcription from a variety of viral and cellular genes by binding to one or more "GC box" recognition elements (containing a hexanuclear core GGGCGG) within the 5' flanking promoter sequences through the use of three "zinc-finger" domains. In general, DNA binding surfaces are designed to have a highly defined preference for their cognate DNA binding site; Sp l is unique among transcription factors identified to date in that it recognizes a host of transcription activating binding sites that can be classified as either high, medium or low affinity. We are therefore quite interested in examining the binding of Sp1 to "GC box" sequences in order to define those factors responsible for this unusually promiscuous sequence recognition ability. The Spl system is amenable to detailed examination owing to localization of its DNA binding properties to the "zinc-finger" domain. This proposal describes an approach that will enable us to exploit the relatively compact "zinc-finger" motif to define those structural factors responsible for the recognition diversity of Sp 1. Our objectives are: ( 1) to express short soluble Sp 1 fragments that contain the three "zinc finger" domains, (2) to test the ability of these fragments to duplicate the natural binding properties of intact Spl by band shift assays, (3) to express and purify large quantities of those peptides that exhibit Spl binding ability, (4) to quantitatively characterize the interactions of these fragments with duplex oligonucleotides by molecular biological (band shift assays, competition assays, mutagenesis studies) and biophysical techniques (calorimetry, NMR) in order to define the chemical/structural basis for the observed diversity in DNA binding, and (5) to determine the solution structures of the active Spl fragments by NMR spectroscopic techniques. Our long term objective is to characterize the solution structures of a number of Spl -DNA complexes using a series of high and medium affinity DNA recognition sequences. Such data will not only contribute to our understanding at the molecular level of how "zinc-finger" domains are used in molecular recognition but also how this major structural motif is able to recognize a highly variable set of DNA sequences.