PROTEIN BINDING DOMAINS ON EUKARYOTIC 5S RRNA AND RDNA

真核 5S RRNA 和 RDNA 上的蛋白质结合域

基本信息

批准号：
2179199
负责人：
PAUL William HUBER
金额：
$ 18.5万
依托单位：
UNIVERSITY OF NOTRE DAME
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-06-01 至 1996-06-30
项目状态：
已结题

项目摘要

Protein-nucleic acid interactions are the fundamental basis for the control of genetic expression. Xenopus laevis contains two 5S rRNA multigene families that provide a simple, tractable model for the de- velopmental regulation of transcription by trans-acting factors. Our long term goal is to understand the temporal expression of these two gene families as a model for more complex developmental systems. The syn- thesis of 5S rRNA is primarily mediated by the zinc finger transcription factor TFIIIA which has the distinctive ability to bind to the 5S rRNA gene as a positive regulator of transcription and to the gene's transcript, 5S rRNA, to form a storage particle for the latter nucleic acid. We have concentrated on the ability of TFIIIA to bind to both nucleic acids since this is fundamental to the regulatory activity of the protein and because it provides a unique problem with regard to protein-nucleic acid recognition. The two TFIIIA-nucleic acid complexes have been crosslinked by UV irradiation. The sites of adduct formation on the protein will be determined in order to assess the relative importance of the nine fingers for binding to the different nucleic acids and whether the linker sequences that connect the fingers form important contacts to either nucleic acid. A combination of molecular biology and organic chemistry will be used to introduce a metal-binding amino acid analogue at specific sites within TFIIIA. The precise alignment of the factor along both the gene and 5S RNA will be mapped by exploiting the nucleic acid cleavage activity of the appended metal. These modified forms of the factor also will be used to study its interaction with the gene in the dynamic conditions of transcription when RNA polymerase moves through, but does not disrupt the transcription complex. Transition metal complexes that bind to RNA on the basis of shape complementarity will be used to delineate the tertiary organization of the molecule. Using specific mutants of the RNA, we will determine whether there is a correlation between the identified structures and recognition by TFIIIA. We have detected an interaction between ribosomes and the oocyte 5S rRNA genes. We will characterize this interaction to ascertain whether it is specific. We will determine whether it is related to the specific destabilization of transcription complexes on oocyte genes that occurs at meiosis. Chemical nucleases were employed to analyze the TFIIIA.5S rRNA particle. A similar analysis of the complex of ribosomal protein L5 with 5S rRNA will be undertaken. This affords the opportunity to examine the binding of two proteins, that exhibit no sequence identity, to congruent sites on an RNA molecule.

蛋白质与核酸的相互作用是蛋白质与核酸相互作用的基础基因表达的控制。非洲爪蟾含有两个 5S rRNA 多基因家族为解构提供了一个简单、易处理的模型反式作用因子对转录的发育调节。我们的长期目标是了解这两个基因的时间表达家庭作为更复杂的发展系统的模型。同步 5S rRNA的论文主要由锌指转录介导因子 TFIIIA 具有与 5S rRNA 结合的独特能力基因作为转录的正调节因子并影响基因的转录物，5S rRNA，形成后一个核酸的存储颗粒酸。我们专注于 TFIIIA 结合两者的能力核酸，因为这是调节活性的基础蛋白质，因为它提供了一个独特的问题蛋白质-核酸识别。两个 TFIIIA-核酸复合物已通过 UV 交联辐照。蛋白质上加合物形成的位点是确定以评估九个手指的相对重要性用于结合不同的核酸以及接头是否连接手指的序列形成重要的接触核酸。分子生物学和有机化学的结合将被用来在特定位点引入金属结合氨基酸类似物 TFIIIA。因子沿基因和 5S 的精确排列 RNA将通过利用核酸切割活性来绘制附加金属。该因子的这些修改形式也将被使用研究其在动态条件下与基因的相互作用当RNA聚合酶移动时转录，但不破坏转录复合物。根据形状与 RNA 结合的过渡金属配合物互补性将被用来描述第三级组织分子。使用 RNA 的特定突变体，我们将确定所识别的结构之间是否存在相关性获得 TFIIIA 的认可。我们检测到核糖体和卵母细胞 5S rRNA 之间的相互作用基因。我们将描述这种相互作用以确定它是否是具体的。我们将确定是否与具体情况有关卵母细胞基因转录复合物的不稳定发生在减数分裂。使用化学核酸酶分析 TFIIIA.5S rRNA 颗粒。核糖体蛋白 L5 与 5S rRNA 复合物的类似分析将进行。这提供了检查绑定的机会两个蛋白质，没有表现出序列同一性，到一致的位点 RNA分子。