INTERACTIONS OF THE A1 AND A2 PROTEINS OF S CEREVISIAE

这项建议的广泛、长期目标是理解 相互承认的基本原则 转录因子A1和Alpha2相互作用和相互作用 目标是DNA站点。这两种调节蛋白是最重要的 酿酒酵母细胞类型的决定因素。在二倍体细胞中 存在A1和Alpha2两种类型，并形成杂二聚体 特别是抑制一组单倍体特有的基因。两种蛋白质 含有一个使用螺旋-旋转-螺旋的同源DNA结合基序 单元和用于DNA识别的灵活的氨基末端臂。尽管 对这一蛋白质家族的详细结构研究，我们仍然没有 了解同源结构域的专一性背后的细节- DNA结合作用和异源二聚作用对DNA的影响 同源域结构和DNA识别事件。 提出的研究的具体目标是：(1)确定 功能DNA结合的详细溶液结构和 A1和Alpha2蛋白的异源二聚结构域，均在 溶液，并作为杂二聚体复合体的一部分。使用核磁 共振(核磁共振)谱，固有的结构差异 两种蛋白质的同源结构域之间的关系，特别是 在A1同源结构域上可以研究异源二聚作用。 这些结构与最近发表的三元结构的比较 复合体将揭示蛋白质必须发生的结构变化 形成一种特定的复合体。(2)主干和主干的动力 A1和Alpha2同源域中的侧链原子--以自由状态 形式，作为异源二聚体结合，并结合到它们的目标DNA位点- 将使用异核核磁共振方法进行表征。松驰 参数测量将突出灵活性方面的差异 在两个同源结构域蛋白之间作为特异性转录 复合体是逐渐形成的。(3)与之相关的能量学 将使用量热法对复杂的形成进行表征。 这些研究将提供一种获得热力学信息的方法 论同源结构域上结构和动态变化的重要性 形成了复合体。最后，(4)突变蛋白和不同的结构 这些蛋白质的研究为扩展这些研究提供了机会。 突变体和不同突变体的结构、动力学和热力学研究 A1和Alpha2同源结构域的构建将使我们能够深入了解 个体相互作用的重要性和突变的影响 以及周围结构对识别过程的影响。 目前，数百种同源结构域蛋白已在 真核生物从酵母到人类并在发育中发挥关键作用 分化和细胞生长过程。尤其是A1 蛋白质与嵌合体的Pbx1同源结构域密切相关 融合蛋白与前B细胞急性淋巴母细胞 白血病。因此，这些研究有助于加深对 除了其他正常的生长和发育外，肿瘤的发生 流程。

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.