SPECIFICITY OF DNA BINDING BY ZINC FINGER PROTEIN

转录因子IIIA（TFIIIA）是锌指核酸的原型 酸结合蛋白，是转录的特异性要求， 两栖动物爪蟾的5S RNA基因。 虽然这种蛋白质已经 在DNA结合和转录活性方面进行了广泛的研究， 关于这种重要蛋白质的详细结构信息仍然缺乏- DNA复合体。 尝试在结晶的全长，九锌 手指蛋白或蛋白质-DNA复合物尚未成功。 从 TFIIIA的cDNA克隆的衍生物，我们将通过 在细菌中的过表达，TFIIIA蛋白的截短形式。 这些 将纯化多肽并测试DNA结合活性。 从 通过这些研究，我们将确定锌指的最小数量， 序列特异性结合所需的。 的热力学性质 将测定这些络合物（缔合常数和半衰期）。 我们将使用阿托伐他汀定向定点突变来改变 TFIIIA多肽关键锌指中的特定氨基酸 并研究这些突变对DNA结合的影响。 突变将被 在多肽的指状区和接头区中产生。 我们将使用 短的双链脱氧核糖-寡核苷酸来定义最小的DNA 高亲和力DNA序列特异性结合所需的序列。 的 将评估特定突变对多肽结合的影响。 TFIIIA多肽和与这些多肽的突变多肽复合物 将制备足够量的寡核苷酸， 三维高分辨率核磁共振波谱。 我们 将使用紫外线交联与光敏合成DNA探针，以映射 TFIIIA的氨基酸残基参与特异性DNA相互作用。 这些 研究将扩展我们对TFIIIA特定相互作用的认识， 与5S RNA基因，并提供洞察的一般机制， 蛋白质-DNA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.