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

蛋白质结合域--真核五S RRNA

• 批准号: 3466187
• 负责人: PAUL William HUBER
• 金额: $ 8.68万
• 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 1992-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466187
• 关键词: DNA binding protein; 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.

爪蟾 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活性介导 因子 IIIA (TFIIIA)。 这种蛋白质具有独特的结合能力 5S 基因及其转录物 5S rRNA。 TFIIIA 以及整个 控制卵母细胞和体细胞 5S 活性的蛋白质集合 基因是研究基因发育调控的原型 表达和决心。 5S rRNA 上 TFIIIA 结合位点的鉴定使我们能够 提出该蛋白质利用类似的接触来结合 核酸。 我们将以两种方式测试该提案。 使用圆形 二色性光谱我们将确定 DNA 的构象 TFIIIA 与 5S 基因结合。 如果蛋白质与 两个核酸相似，则 DNA 必须位于 A 中 构象以形成与 5S rRNA 相当的结构。 我们将 进行一系列广泛的定点诱变实验 改变基因和转录本。 我们将确定 特定变化是否对 TFIIIA 的结合具有类似的影响 至两种核酸。 TFIIIA 5S rRNA 上的结合位点与 TFIIIA 的结合位点非常接近 核糖体蛋白L5。 这表明这两种蛋白质可能相关。 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相互作用 蛋白质及其同源核酸。 我们将测试同源性 通过免疫化学技术分析蛋白质。 我们将准备多克隆 含有核酸的 TFIIIA 蛋白水解片段的抗体 蛋白质的酸结合域。 我们将确定是否 抗血清与 L5 发生交叉反应。 如果结果是肯定的，我们将使用 抗血清将核酸结合结构域定位在 L5 内。 TFIIIA 的独特属性使我们能够做出某些无价的 比较：两种核酸的结构之间的比较 被该蛋白质识别，以及识别该蛋白质的两个蛋白质之间 5S rRNA 上有相同的结合位点。