Transcriptional Activator Complex of the Mammalian Circadian Clock

项目摘要 昼夜节律钟是内源性的~24小时振荡器，其驱动生物钟的每日节律。 生物过程。在哺乳动物中，生物钟存在于大脑中， 外周组织分布式时钟共同构成了基本的计时系统， 调节生理代谢和行为 哺乳动物的生物钟建立在转录负反馈环上， 在分子水平上产生昼夜节律。转录因子CLOCK-BMAL 1 是这个反馈回路的核心，是生物钟的核心积极驱动器 转录。人们认为CLOCK-BMAL 1在体内作为异源二聚体发挥作用，但我们 已经表明，在哺乳动物组织的核提取物中， 在昼夜节律周期的转录激活阶段，约800-kDa蛋白复合物， 远大于~170-kDa异二聚体。我们初步的生化分析，质谱分析， 和单粒子电子显微镜的结果出人意料地表明， 复合物完全或大部分由组装成 低聚结构。 这些观察结果告诉我们， 生物钟转录激活因子仍有待发现，这是我们研究中一个基本空白 认识这个应用程序的目标是分析组成，功能，和三个- 神秘的~800-kDa CLOCK-BMAL 1复合物的三维结构。如果成功， 项目提供了扩展和深化我们的昼夜节律钟转录的知识，可能 达到原子级的分辨率 对生物钟理解的进步将对我们的研究产生重要影响。 行为和生理调节的知识，以及对人类健康和 疾病生物钟缺陷导致广泛的生理功能障碍， 睡眠-觉醒周期紊乱、代谢综合征和癌症发病率增加。那里 也是生物钟功能障碍和情绪障碍，包括双相情感障碍之间的联系。的 拟议的调查旨在提供深入了解生物的分子机制， 定时系统对健康至关重要。

Project Summary Circadian clocks are endogenous ~24-hour oscillators that drive daily rhythms of biological processes. In mammals, circadian clocks are found in the brain and in most peripheral tissues. Together, the distributed clocks constitute the basic timing system that regulates physiology, metabolism, and behavior. The mammalian circadian clock is built on a transcriptional negative feedback loop that generates circadian rhythms at the molecular level. The transcription factor CLOCK-BMAL1 is at the heart of this feedback loop, acting as the core positive driver of circadian clock transcription. CLOCK-BMAL1 has been assumed to act in vivo as a heterodimer, but we have shown that in nuclear extracts from mammalian tissues it is exclusively embedded in an ~800-kDa protein complex during the transcriptional activation phase of the circadian cycle, far larger than the ~170-kDa heterodimer. Our preliminary biochemical, mass spectrometry, and single-particle electron microscopy results together unexpectedly suggest that this complex consists entirely or mostly of multiple CLOCK-BMAL1 heterodimers assembled into an oligomeric structure. These observations tell us that the full molecular nature and functional properties of the circadian clock transcriptional activator remain to be discovered, a fundamental gap in our understanding. The goal of this application is to analyze the composition, function, and three- dimensional structure of the enigmatic ~800-kDa CLOCK-BMAL1 complex. If successful, the project offers to extend and deepen our knowledge of circadian clock transcription, possibly to an atomic level of resolution. Advances in understanding the circadian clock will have important implications for our knowledge of the regulation of behavior and physiology, as well as for human health and disease. Circadian clock defects result in broad physiological dysfunction, producing disrupted sleep-wake cycles, metabolic syndrome, and increased cancer incidence. There are also links between clock dysfunction and mood disorders, including bipolar disorder. The proposed investigation aims to provide insights into the molecular machinery of the biological timing system essential for health.