STRUCTURAL CHARACTERIZATION OF SP1-DNA COMPLEXES

RNA聚合酶II合成信使RNA需要相互作用 大量的辅助转录因子识别和 与真核生物上游的特定启动子DNA序列结合 基因。这些转录因子调节着 通过组装和接合以时间顺序的方式转录 活性转录复合体。为了了解详细的信息 转录因子所起的作用，已经做出了努力 分离重组转录所必需的因素 体外活性。这些实验已经导致了 其中一种的鉴定、纯化和性质研究 启动子特异性转录因子，Sp L，来自HeLa细胞。SP L 通过以下方式增强各种病毒和细胞基因的转录 绑定到一个或多个“GC box”识别元素(包含 六核核心GGGCGG)位于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定义的 偏爱其同源DNA结合位点；Sp L是 到目前为止发现的转录因子是因为它识别一系列 转录激活结合位点，可归类为 高亲和力、中亲和力或低亲和力。因此，我们非常有兴趣 检查Sp1与“GC box”序列的结合，以确定 这些因素导致了这种不寻常的混杂序列 识别能力。SPL系统可以接受详细检查 由于其与“锌指”的DNA结合特性的局部化 域。这项提案描述了一种方法，使我们能够 利用相对紧凑的“锌指”主题来定义这些 Sp-1识别多样性的结构性因素。 我们的目标是：(1)表达可溶短的Sp-1片段 包含三个锌指结构域，(2)测试这些结构域的能力 复制完整SPL的自然结合特性的片段 条带移位分析，(3)大量表达和纯化 显示SPL结合能力的多肽，(4)定量结合 表征这些片段与双链的相互作用 通过分子生物学的寡核苷酸(带移位分析、竞争 化验、诱变研究)和生物物理技术(量热法、 核磁共振)，以确定观察到的化学/结构基础 DNA结合的多样性，以及(5)确定其溶液结构 用核磁共振波谱技术测定活性SPL片段。 我们的长期目标是刻画一个 使用一系列高亲和力和中亲和力的SPL-DNA复合体的数量 DNA识别序列。这些数据不仅将有助于我们的 在分子水平上理解“锌指”结构域是如何 用于分子识别，以及这个主要的结构基序是如何 能够识别一组高度可变的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.