INTERACTIONS OF THE A1 AND A2 PROTEINS OF S CEREVISIAE

酿酒酵母 A1 和 A2 蛋白的相互作用

• 批准号: 6164789
• 负责人: Griselda Hernandez
• 金额: $ 10.01万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6164789
• 关键词: DNA binding protein; Saccharomyces cerevisiae; calorimetry; cell type; nuclear magnetic resonance spectroscopy; nucleic acid structure; protein structure function; structural biology; thermodynamics; transcription factor

The broad, long term objective of this proposal is to understand the fundamental principles underlying the recognition between the transcriptional factors, a1 and alpha2, for each other and for their target DNA sites. These two regulatory proteins are the prime determinants of cell type in Saccharomyces cerevisiae. In diploid cell types, both a1 and alpha2 are present and form a heterodimer to specifically repress a set of haploid-specific genes. Both proteins contain a homeoJomain DNA-binding motif which uses a helix-turn-helix unit and a flexible amino-terminal arm for DNA recognition. Despite detailed structural studies of this family of proteins, we still do not understand the details underlying the specificity of the homeodomain- DNA association and the effects of heterodimerization on the homeodomain structure and the DNA recognition event. The specific aims of the research proposed are: (1) to determine detailed solution structures of the functional DNA-binding and heterodimerizing domains of the a1 and alpha2 proteins, both free in solution and as part of the heterodimer complex. Using nuclear magnetic resonance (NMR) spectroscopy, the inherent structural differences between the homeodomains of the two proteins and especially the changes in the a1 homeodomain upon heterodimerization can be studied. Comparison of these structures with the recently published ternary complex will reveal the structural changes that the proteins must undergo to form a specific complex. (2) The dynamics of backbone and sidechain atoms in the a1 and alpha2 homeodomains - in their free forms, bound as the heterodimer and bound to their target DNA sites - will be characterized using heteronuclear NMR methods. Relaxation parameter measurements will highlight the differences in flexibility between the two homeodomain proteins as the specific transcriptional complex is formed incrementally. (3) The energetics associated with complex formation will be characterized using calorimetric methods. These studies will provide a means to gain thermodynamic information on the importance of structural and dynamic changes on the homeodomain complexes formed. Lastly, (4) mutant proteins and different constructs of the proteins offer an opportunity to extend these studies. Structural, dynamic and thermodynamic studies of mutants and differing constructs of the a1 and alpha2 homeodomains will allow insight into the importance of individual interactions and the effects of mutations and surrounding structure on the recognition processes. Currently hundreds of homeodomain proteins have been identified in eukaryotes from yeast to human and play critical roles in development differentiation and cell growth processes. In particular, the a1 protein is related closely to the pbx1 homeodomain of the chimeric fusion protein associated with pre-B cell acute lymphoblastic leukemias. Thus these studies contribute to an increased understanding of oncogenesis in addition to other normal growth and development processes.

该提议的广泛，长期目标是了解 认识的基本原则 转录因素A1和Alpha2彼此及其 目标DNA位点。这两种调节蛋白是主要的 酿酒酵母中细胞类型的决定因素。在二倍体细胞中 类型，A1和Alpha2都存在并形成异二聚体 特别抑制一组单倍体特异性基因。两种蛋白质 包含使用螺旋转螺螺旋的主角DNA结合图案 单位和柔性氨基末端组，用于DNA识别。尽管 该蛋白质家族的详细结构研究，我们仍然没有 了解同源域特异性的细节 - DNA关联和异二聚化对 同源域结构和DNA识别事件。 提出的研究的具体目的是：（1）确定 功能性DNA结合的详细溶液结构和 A1和Alpha2蛋白的异二聚体结构域，都在 解决方案，作为异二聚体复合物的一部分。使用核磁 共振（NMR）光谱，固有的结构差异 在两种蛋白质的同源域之间，尤其是变化之间 在异二聚化时，可以研究A1同源域中。 这些结构与最近发表的三元 复杂将揭示蛋白质必须的结构变化 要形成特定的复合物。 （2）骨干的动力学和 A1和alpha2同源域中的Sidechain原子 - 免费 形式，作为异二聚体结合并与其靶DNA位点结合 - 将使用异核NMR方法进行表征。松弛 参数测量将突出灵活性的差异 在两种同源域蛋白作为特定转录之间 复合物是逐渐形成的。 （3）与 复合形成将使用量热法进行表征。 这些研究将提供一种获得热力学信息的方法 关于同源域的结构和动态变化的重要性 形成复合物。最后，（4）突变蛋白和不同的构建体 蛋白质的大量提供了扩展这些研究的机会。 突变体和不同的结构，动态和热力学研究 A1和Alpha2同源域的结构将使您深入了解 单个相互作用的重要性和突变的影响 以及识别过程中的周围结构。 目前已经确定了数百种同源域蛋白 从酵母到人类的真核生物，在发展中起关键作用 分化和细胞生长过程。特别是A1 蛋白质与嵌合的PBX1同源域紧密相关 与前B细胞急性淋巴细胞相关的融合蛋白 白血病。因此，这些研究有助于增加理解 除其他正常生长和发育外 过程。