DNA BINDING OF RECOMBINANT TFII D PROTEINS

描述(改编自申请者摘要)：基因调控 表达对细胞的反应能力至关重要 对它的环境，控制细胞生长和过程 发展。某些基因的异常表达可能导致不受调控 细胞生长和癌症的发展。在大多数情况下，基因表达 在信使核糖核酸合成水平上受到调控。申请者正在研究 RNA聚合酶II所需的通用转录因子 在所有启动子位置准确启动信使核糖核酸的合成。第一 在体外与DNA模板结合的一般因素中，有一种是TATA盒 转录因子，TFIID。TFIID的绑定启动了订单 转录机器的其余部件的组装。 因为从哺乳动物中提纯TFIID活性很困难 与其他后生动物细胞一样，人们对其生化特性知之甚少。 最近，编码酵母TFIID蛋白的基因已经被克隆并 这使得TFIID蛋白的cDNA克隆能够从其他 包括人类在内的物种。克隆的TFIID蛋白在大肠杆菌中的表达 细菌提供足够量的活性TFIID用于详细的 这一重要转录本的生化特性分析 因素。 这里提出的研究将定义形成 一个具有功能的TFIID蛋白-DNA复合体。平衡解离 常数，结合和解离的速率常数为 用硝酸纤维滤膜结合试验和TFIID蛋白测定 从重组大肠杆菌中提纯。DNA结合的活化能 酵母TFIID的野生型和N端缺失突变体将 测量以确定N末端是否调节TFIID-DNA 有约束力的。通过TFIID进行特定绑定的序列要求如下 使用包含变异序列的寡核苷酸池进行检查 替换一致的TATA框元素或替换序列 通常位于共识塔塔盒子元素的侧翼。寡核苷酸 与TFIID紧密结合将在过滤后被分离 用聚合酶链式反应进行扩增。重复循环 选择性结合和扩增将识别重要的残基 用于TFIID的序列特定结合。因为转录的过程 本质上是一个不对称的过程(聚合酶II分子转录 仅在转录起始位置的一个方向上)，并且由于 TFIID的结合启动了转录复合体的组装，它是 不对称定向的TFIID蛋白可能会使 形成不对称的复合体。TFIID绑定到一个 塔塔箱体元件将通过对紧密接触的分析确定 这种蛋白质与DNA结合，通过使用特定的N-末端 多肽抗体。

DESCRIPTION (Adapted from Applicant's Abstract): The regulation of gene expression is of central importance to the ability of the cell to respond to its environment, to control cell growth and to the process of development. Aberrant expression of some genes may lead to unregulated cell growth and the development of cancer. In most cases, gene expression is regulated at the level of mRNA synthesis. The applicant is studying the general transcription factors which are required by RNA polymerase II for the accurate initiation of mRNA synthesis at all promoter sites. The first of the general factors to bind to a DNA template in vitro is the TATA box transcription factor, TFIID. Binding of TFIID initiates the ordered assembly of the remaining components of the transcriptional machinery. Because of the difficulty in purifying the TFIID activity from mammalian and other metazoan cells, little is known about its biochemical properties. Recently, the gene encoding the yeast TFIID protein has been cloned and this allowed the isolation of cDNA clones of TFIID proteins from other species including human. Expression of the cloned TFIID proteins in bacteria provides sufficient quantities of active TFIID for detailed analyses of the biochemical properties of this important transcription factor. The studies proposed here will define the requirements for the formation of a functional TFIID protein-DNA complex. The equilibrium dissociation constant and the rate constants for binding and dissociation will be determined using a nitrocellulose filter binding assay and TFIID proteins purified from recombinant E. coli. The activation energy for DNA binding by wild type and an N-terminal deletion mutant of yeast TFIID will be measured in order to determine whether the N-terminus regulates TFIID-DNA binding. The sequence requirements for specific binding by TFIID will be examined using pools of oligonucleotide containing variant sequences in place of either a consensus TATA box element or in place of the sequences which normally flank a consensus TATA box element. Oligonucleotide which bind tightly to TFIID will be isolated following filtration through nitrocellulose and amplified by polymerase chain reaction. Repeated cycles of selective binding and amplification will identify the residues important for sequence specific binding by TFIID. Since the process of transcription is inherently an asymmetric process (the polymerase II molecule transcribes in only one direction from the start site of transcription) and since the binding of TFIID initiates the assembly of the transcription complex, it is possible that an asymmetrically oriented TFIID protein could nucleate the formation of an asymmetric complex. The symmetry of TFIID binding to a TATA box element will be determined by the analysis of the close contacts the protein makes with the DNA and by the use of a specific N-terminal peptide antibody.