SPECIFICITY OF DNA BINDING BY ZINC FINGER PROTEIN

转录因子IIIA(TFIIIA)是锌指核的原型 是一种酸结合蛋白，是转录 非洲爪哇两栖动物中的5S RNA基因。尽管这种蛋白质已经 在DNA结合和转录活性方面进行了广泛的研究， 关于这一重要蛋白质的详细结构信息仍然缺乏- DNA复合体。尝试结晶的全长，九锌 手指蛋白或蛋白质-DNA复合体还没有成功。从… TFIIIA的cDNA克隆的衍生品，我们将通过 在细菌中过表达，截短版本的TFIIIA蛋白。这些 多肽将被提纯并测试DNA结合活性。从… 在这些研究中，我们将确定锌指的最小数量 序列特定绑定所必需的。分子的热力学性质 这些络合物(缔合常数和半衰期)将被确定。 我们将使用寡核苷酸导向的定点突变来改变 TFIIIA多肽临界锌指中的特定氨基酸 并研究这些突变对DNA结合的影响。突变将是 在多肽的指状区和连接区制造。我们将使用 用短双链脱氧核糖核酸确定最小DNA 高亲和力DNA序列特异性结合所需的序列。这个 将评估特定突变对多肽结合的影响。 TFIIIA多肽及其突变型多肽复合体 将制备足够数量的寡核苷酸，供两个 三维高分辨率核磁共振波谱学。我们 将使用紫外光交联和光活性合成DNA探针来绘制 TFIIIA的氨基酸残基参与了特异的DNA相互作用。这些 研究将扩大我们对TFIIIA具体相互作用的了解 并提供了对5S RNA基因的一般机制的深入了解 蛋白质与DNA的相互作用。

Transcription factor IIIA (TFIIIA) is the prototype zinc finger nucleic acid-binding protein and is specifically required for transcription of the 5S RNA genes in the amphibian Xenopus. Although this protein has been studied extensively in terms of DNA binding and transcriptional activity, detailed structural information is still lacking on this important protein- DNA complex. Attempts at crystallization of the full length, nine zinc finger protein or the protein-DNA complex have not been successful. From derivatives of the cDNA clone for TFIIIA, we will produce, by overexpression in bacteria, truncated versions of TFIIIA protein. These polypeptides will be purified and tested for DNA binding activity. From these studies, we will determine the minimum number of zinc fingers required for sequence-specific binding. The thermodynamic properties of these complexes (association constants and half-lives) will be determined. We will use oligonucleotide-directed site-specific mutagenesis to alter specific amino acids in the critical zinc fingers of TFIIIA polypeptides and study the effects of these mutations on DNA binding. Mutations will be made in both finger and linker regions of the polypeptides. We will use short double stranded deoxyribo-oligonucleotides to define the minimal DNA sequences required for high affinity DNA sequence-specific binding. The effects of specific mutations on polypeptide binding will be assessed. TFIIIA polypeptide and mutant polypeptide complexes with these oligonucleotides will be prepared in sufficient quantity for two dimensional high resolution nuclear magnetic resonance spectroscopy. We will use UV-crosslinking with photoactive synthetic DNA probes to map the amino acid residues of TFIIIA involved in specific DNA interactions. These studies will extend our knowledge of the specific interaction of TFIIIA with the 5S RNA genes and provide insights into the general mechanisms of protein-DNA interactions.