Mechanistic Studies of Progesterone Receptor Function

超出所提供的空间。本研究的长期目标是阐明真核生物基因调控的分子机制。重点是人类孕激素受体（PR）与复杂启动子的协同结合机制，以及激素激动剂和拮抗剂在调节这些反应中的作用。进一步的目标是确定将相关结构转换传播到邻近域的原则。PR以两种功能不同的亚型共存：83 kD a受体和99 kD b受体。这两种同工异构体是相同的，除了b受体在其n端有额外的164个氨基酸。b受体通常作为强激活因子，而a受体通常作为弱激活因子。据推测，这种差异是由于b受体在pr调控的启动子处协同结合的能力引起的。从机制上讲，b -唯一残基对受体的其余部分施加激素依赖的构象约束。这种约束导致了PR结构和稳定性的变化，这些变化可能包括戏剧性的无序转换，从而导致DNA的合作结合。有人提出拮抗剂的作用是通过稳定PR激素结合域内的无效构象来解耦这些联系。这一假设和潜在的机制将通过以下研究进行检验：目的1 -在存在和不存在黄体酮激动剂和拮抗剂的情况下，两种异构体的自组装能量学将使用分析性超离心确定。目的2 -将使用定量dna酶足迹确定每个PR异构体与多位点小鼠乳腺肿瘤病毒启动子相互作用的严格热力学分析。目标3 -将利用羟基自由基蛋白水解足迹、CD光谱和微量热法绘制与配体和dna结合相关的异构体结构和稳定性的变化。网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. The long-term goal of this research is to elucidate the molecular mechanisms underlying eukaryotic gene regulation. Focus is centered on the mechanisms by which human progesterone receptors (PR) cooperatively bind to complex promoters, and the role of hormone agonists and antagonists in regulating these reactions. A further goat is to determine the principles by which the associated structural transitions are propagated to neighboring domains. PR co-exist as two functionally distinct isoforms: an 83 kD A-receptor and a 99 kD B-receptor. The two isoforms are identical except that the B-receptor has an additional 164 amino acids at its N-terminus. The B-receptor often functions as a strong transcriptional activator while the A-receptor generally acts as a weak activator. It is hypothesized that this difference arises through the ability of the B-receptor to bind cooperatively at PR-regulated promoters. Mechanistically, the B-unique residues impose a hormone-dependent conformational constraint upon the remainder of the receptor. This constraint causes changes in PR structure and stability- changes that can include dramatic disorder-order transitions - resulting in cooperative DNA binding. It is proposed that a role of antagonists is to decouple these linkages by stabilizing ineffective conformations within the PR hormone-binding domain. This hypothesis and the underlying mechanism will be examined by carrying out the following studies: Aim 1 - The energetics of self-assembly for both isoforms in the presence and absence of progestin agonists and antagonists will be determined using analytical ultracentrifugation. Aim 2 - A rigorous thermodynamic analysis of the interactions of each PR isoform with the multi-site mouse mammary tumor virus promoter will be determined using quantitative DNAse footprinting. Aim 3 - The changes in isoform structure and stability associated with ligand and DNA-binding will be mapped using hydroxyl radical proteolytic footprinting, CD spectroscopy and microcalorimetry. PERFORMANCE SITE ========================================Section End===========================================