DNA BINDING ACTIVITY OF RECOMBINANT TFII D PROTEINS

描述（改编自申请人的摘要）：基因的调节 表达对于细胞应答的能力至关重要 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的 细胞生长和癌症的发展。 在大多数情况下，基因表达 在mRNA合成水平上受到调控。 申请人正在研究 RNA聚合酶II所需的一般转录因子， 在所有启动子位点精确启动mRNA合成。 第一 在体外与DNA模板结合的一般因子是TATA盒 转录因子TFIID。 TFIID的绑定启动有序的 转录机器的其余组件的组装。 由于难以从哺乳动物中纯化TFIID活性， 和其他后生动物细胞，很少有人知道它的生化特性。 最近，已经克隆了编码酵母TFIID蛋白的基因， 这允许从其它细胞中分离TFIID蛋白的cDNA克隆， 包括人类在内的物种。 克隆的TFIID蛋白在大肠杆菌中的表达 细菌提供了足够数量的活性TFIID， 分析这一重要转录的生化特性， 因子 这里提出的研究将确定形成 功能性TFIID蛋白-DNA复合物。 平衡解离 常数，结合和解离的速率常数为 使用硝酸纤维素滤膜结合试验和TFIID蛋白测定 从重组E.杆菌 DNA结合的活化能 通过野生型和酵母TFIID的N-末端缺失突变体， 测量以确定N-末端是否调节TFIID-DNA 约束力 通过TFIID特异性结合的序列要求将是 使用含有变异序列的寡核苷酸池进行检查， 替换共有TATA盒元件或替换序列 其通常位于共有TATA盒元件的侧翼。 寡核苷酸 与TFIID紧密结合，将在过滤后分离， 硝酸纤维素，并通过聚合酶链反应扩增。 重复循环 的选择性结合和扩增将确定残基的重要性， 用于通过TFIID的序列特异性结合。 由于转录的过程 本质上是一个不对称过程（聚合酶II分子转录 从转录起始位点仅一个方向），并且由于 TFIID的结合启动转录复合物的组装， 不对称取向的TFIID蛋白可能使 形成不对称复合物。 TFIID结合到a TATA盒元素将通过分析密切接触者来确定 蛋白质与DNA结合，并通过使用特定的N-末端， 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.