Regulation of RNA Polymerase II by the Ess1 Prolyl Isomerase

项目摘要 长期的目标是了解肽基-脯氨基顺式/反式 异构酶(PPIase)是调节基因活性的分子开关。 PPIase催化环氨基前面的多肽键的异构化 酸性脯氨酸，引起构象变化，促进新合成的 并通过改变成熟蛋白质的活性或蛋白质来调节成熟蛋白质的活性- 蛋白质的相互作用。PPIase存在于所有生物体中，最为人所知的是它的靶标。 免疫抑制药物。然而，对它们在细胞中的正常功能知之甚少。 我们研究了一种名为Ess1的PPIase，它是酿酒酵母生长所必需的。 Ess1及其人类同源基因Pin1(可以在酵母中替代Ess1)与 转录调控与有丝分裂细胞周期调控。在病原真菌中，念珠菌 白色念珠菌和新生隐球菌，Ess1对毒力是必不可少的。在人类身上， Pin1的错误表达可能导致癌症和神经退行性疾病。这些 研究结果明确了脯氨基异构化对细胞功能的重要性。 拟议研究的目标是了解Ess1通过什么机制 识别和调节真核细胞中的RNA聚合酶II(RNAPII)。从这里开始工作 实验室表明，Ess1结合并异构化了 RNAPII大亚基的羧基末端结构域(CTD)。Ess1通过抛出 因此，构象“脯氨酸开关”在确定所谓的“CTD”中起着不可或缺的作用 帮助协调蛋白质辅助因子的招募到转录的RNAPII的密码 很复杂。ESS1诱导的CTD构象变化可能对 转录周期中的多个步骤。 Aim1的目标是理解Ess1如何“读取CTD代码”，这是关键的第一步 在其对RNAPII转录周期的调控中。确定Ess1如何识别和 将CTD结合结构、生化、生物物理和体内研究将 被利用。这一结果将对理解哺乳动物的直系同源基因(Pin1)具有重要意义。 识别RNAPII和各种其他底物。这些信息也将对以下方面有用 努力开发抗真菌抑制剂(Ess1)或抗癌药物(Pin1)。 AIM2的目标是了解Ess1是如何通过关注它的 在转录延伸中的作用。工作假设是Ess1控制招聘 和/或延伸因子和/或染色质修饰物的活性，从而调节RNAPII 伸长率。这将使用生化和基因组方法进行测试。这些结果 将是重要的，主要有两个原因。首先，它们将帮助我们了解非共价态 脯氨酸开关(相对于共价修饰，如磷酸化--这已经是一个领域 经过认真研究)规范向CTD招募RNAPII辅助因素。其次，他们将提供帮助 美国了解Ess1是如何调节伸长的，这是一种日益被认识的机制 在基因调控中起着关键作用。 据我们所知，这是唯一的建议，我们是唯一的实验室，正在研究这一角色 这一关键的肽基-脯氨基异构酶的转录。

Project Summary The long-term goal is to understand the mechanism by which peptidyl-prolyl cis/trans isomerases (PPIases) function as molecular switches to regulate gene activity. PPIases catalyze the isomerization of the peptide bond that precedes the cyclic amino acid proline, causing conformational changes that facilitate the folding of newly-synthesized proteins and that regulate the activity of mature proteins by altering their activity or protein- protein interactions. PPIases are found in all organisms, and are best known as the targets of immunosuppressive drugs. However, their normal function in cells is poorly understood. We study a PPIase called Ess1, which is essential for growth in Saccharomyces cerevisiae. Ess1 and its human ortholog, Pin1 (which can substitute for Ess1 in yeast), are implicated in transcription regulation and mitotic cell cycle control. In the pathogenic fungi, Candida albicans and Cryptococcus neoformans, Ess1 is essential for virulence. In humans, misexpression of Pin1 may contribute to cancer and neurodegenerative disease. These findings make clear the importance of prolyl isomerization for cellular function. The goal of the proposed research is to understand the mechanism by which Ess1 recognizes and regulates RNA polymerase II (RNAPII) in eukaryotic cells. Work from this laboratory has shown that Ess1 binds and isomerizes peptidyl-prolyl bonds within the carboxy-terminal domain (CTD) of the large subunit of RNAPII. Ess1 acts by throwing a conformational “proline switch” thus plays an integral role in specifying the so-called “CTD code” that helps coordinate the recruitment of protein co-factors to the transcribing RNAPII complex. Ess1-induced conformational changes in the CTD are likely to be important for multiple steps in the transcription cycle. The goal of Aim1 is to understand how Ess1 “reads the CTD code,” a critical first step in its regulation of the RNAPII transcription cycle. To determine how Ess1 recognizes and binds the CTD a combination of structural, biochemical, biophysical and in vivo studies will be used. The results will be important for understanding how the mammalian ortholog (Pin1) recognizes RNAPII and a variety of other substrates. This information will also be useful for efforts to develop antifungal inhibitors (Ess1) or anti-cancer drugs (Pin1). The goal of Aim2 is to understand how Ess1 “writes the CTD code” by focusing on its role in transcription elongation. The working hypothesis is that Ess1 controls the recruitment and/or activity of elongation factors, and/or chromatin modifiers, thus regulating RNAPII elongation. This will be tested using biochemical and genomic approaches. These results will be important for two main reasons. First, they will help us understand how non-covalent proline switches (versus covalent modification such as phosphorylation – an area already heavily studied) regulate recruitment of RNAPII co-factors to the CTD. Second, they will help us understand how Ess1 regulates elongation, a mechanism that is increasingly recognized as critical in gene regulation. To our knowledge, this is the only proposal, and we are the only laboratory, studying the role of this key peptidyl-prolyl isomerase in transcription.