INTERACTIONS OF THE A1 AND A2 PROTEINS OF S CEREVISIAE

本提案的广泛、长期目标是了解 基本原则， 转录因子，α 1和α 2，对于彼此和对于它们的 靶向DNA位点。这两种调节蛋白是 酿酒酵母中细胞类型的决定因素。二倍体细胞 类型，a1和a2都存在，并形成异二聚体， 特异性抑制一组单倍体特异性基因。两种蛋白质 含有一个同源Jomain DNA结合基序，它使用螺旋-转角-螺旋 单元和用于DNA识别的柔性氨基末端臂。尽管 对这个蛋白质家族的详细结构研究，我们仍然没有 了解同源结构域特异性的细节- DNA缔合和异源二聚体化对细胞凋亡的影响 同源结构域结构和DNA识别事件。 本研究的具体目标是：（1）确定 功能性DNA结合的详细溶液结构， α 1和α 2蛋白的异源二聚化结构域，两者都是游离的， 溶液和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 这两种蛋白质的同源结构域之间， 可以研究在异源二聚化后A1同源结构域中的变化。 比较这些结构与最近发表的三元 复合物将揭示蛋白质必须 形成一种特殊的复合物。(2)主干的动力学和 在α 1和α 2同源结构域中的侧链原子-在它们的游离 形式，作为异二聚体结合并结合到其靶DNA位点- 将使用异相NMR方法表征。放松 参数测量将突出显示灵活性的差异 两个同源结构域蛋白之间的特异性转录 复合体逐渐形成。(3)与之相关的能量 络合物的形成将使用量热法表征。 这些研究将为获得热力学信息提供一种手段 关于同源结构域的结构和动态变化的重要性 复合物形成。最后，（4）突变蛋白和不同的构建体 这些蛋白质的研究为扩展这些研究提供了机会。 突变体的结构、动力学和热力学研究以及不同的 α 1和α 2同源结构域的构建体将允许了解 个体相互作用的重要性和突变的影响 和周围的结构。 目前，已经鉴定了数百种同源结构域蛋白， 从酵母到人类的真核生物，在发育过程中起着关键作用。 分化和细胞生长过程。特别是，A1 蛋白质与嵌合体的pbx 1同源域密切相关， 前B细胞急性淋巴细胞相关融合蛋白 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， 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.