PROTEIN BINDING DOMAINS--EUKARYOTIC FIVE-S RRNA & RDNA

蛋白质结合域--真核五S RRNA

• 批准号: 3466189
• 负责人: PAUL William HUBER
• 金额: $ 9.96万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-06-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466189
• 关键词: DNA binding protein; RNA biosynthesis; Xenopus; antibody; binding proteins; chemical binding; chemical structure function; circular dichroism; conformation; genetic manipulation; genetic transcription; histones; immunochemistry; laboratory rabbit; nucleic acid sequence; nucleic acid structure; point mutation; protein engineering; proteins; ribonucleoproteins; ribosomal DNA; ribosomal RNA; ribosomal proteins; ribosomes; site directed mutagenesis

The developmental control of the expression of Xenopus 5S rRNA genes is mediated by the activitiy of an exceptional protein called transcription factor IIIA (TFIIIA). This protein has the distinctive ability to bind to both the 5S gene and its transcript, 5S rRNA. TFIIIA as well as the whole collection of proteins that control the activity of oocyte and somatic 5S genes are the prototype for the study of developmental regulation of gene expression and determination. The identification of the binding site for TFIIIA on 5S rRNA has led us to propose that the protein utilizes similar contacts in binding to either nucleic acid. We will test this proposal in two ways. Using circular dichroism spectroscopy we will determine the conformation of the DNA when TFIIIA binds to the 5S gene. If the interaction of the protein with the two nucleic acids is similar then the DNA will have to be in the A conformation in order to form a structure comparable to 5S rRna. We will carry out an extensive series of site-directed mutagenesis experiments to make changes in both the gene and the transcript. We will determine whether a particular change has a similar effect on the binding of TFIIIA to both nucleic acids. The binding site on 5S rRNA for TFIIIA closely approximates that for ribosomal protein L5. This suggests that the two proteins may be related; at least there may be homology between their nucleic acid binding domains. We will measure the binding of L5 and TFIIIA to the variant 5S rRNAs synthesized in the mutagenesis experiments to compare the interactions of the proteins with their cognate nucleic acid. We will test the homology of the proteins by immunochemical techniques. We will prepare polyclonal antibodies to a proteolytic fragment of TFIIIA that contains the nucleic acid binding domain of the protein. We will determine whether the antiserum crossreacts with L5. If the result is positive, we will use the antiserum to locate the nucleic acid binding domain within L5. The unique properties of TFIIIA allow us to make certain invaluable comparisons: between the structures of two nucleic acids that are recognized by this protein, and between two proteins that recognize the same binding site on 5S rRNA.

异武5S rRNA基因表达的发育控制是 由一种称为转录的特殊蛋白质的激活介导 因子IIIA（TFIIIA）。 该蛋白具有与之结合的独特能力 5S基因及其转录本5s rRNA。 tfiiia和整体 收集控制卵母细胞和体细胞5s活性的蛋白质 基因是研究基因发育调节的原型 表达和确定。 5S rRNA上TFIIIA的结合位点的识别使我们达到了 提出蛋白质在与任何一个结合中使用类似的接触 核酸。 我们将通过两种方式测试该建议。 使用圆形 二分色谱法我们将确定DNA的构象 TFIIIA与5S基因结合。 如果蛋白质与 两个核酸相似，那么DNA必须在A中 构象以形成与5s rRNA相当的结构。 我们将 进行一系列位置定向的诱变实验 改变基因和转录本。 我们将确定 特定的变化是否对TFIIIA的结合有相似的影响 两种核酸。 TFIIIA的5S rRNA上的结合位点非常接近 核糖体蛋白L5。 这表明两种蛋白质可能是相关的。 至少它们的核酸结合结构域之间可能存在同源性。 我们将测量L5和TFIIIA与变体5S rRNA的结合 在诱变实验中合成以比较 蛋白质及其同源核酸。 我们将测试 免疫化学技术的蛋白质。 我们将准备多克隆 含有核的蛋白水解片段的抗体 蛋白质的酸结合结构域。 我们将确定是否 抗血清与L5交叉反应。 如果结果是积极的，我们将使用 抗血清在L5内定位核酸结合结构域。 TFIIIA的独特属性使我们能够发挥一定的宝贵性 比较：在两个核酸的结构之间 被该蛋白质认识到 在5s rRNA上相同的结合位点。