Transcriptional Activator Complex of the Mammalian Circadian Clock

项目摘要 生物钟是内生的~24小时振荡器，驱动每日的节奏 生物过程。在哺乳动物中，生物钟存在于大脑和大多数 周围组织。分布式时钟共同构成了基本的计时系统， 调节生理、新陈代谢和行为。 哺乳动物的生物钟建立在转录负反馈环上，该环 在分子水平上产生昼夜节律。转录因子Clock-BMAL1 是这个反馈回路的核心，起着生物钟的核心正向驱动作用 抄写。Clock-BMAL1在体内被认为是异源二聚体，但我们 已经表明，在哺乳动物组织的核提取液中，它只嵌入到一个 ~800 kDa蛋白质复合体在昼夜节律的转录激活阶段， 远远大于~170 kDa的杂二聚体。我们的初步生化，质谱学， 而单粒子电子显微镜的结果出人意料地表明这 复合体全部或大部分由多个Clock-BMAL1杂二聚体组装成 一种低聚结构。 这些观察告诉我们，分子的全部性质和功能性质 生物钟转录激活因子仍有待发现，这是我们 理解。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-BMAL1复合体的空间结构。如果成功，则 项目提供了扩展和加深我们对生物钟转录的知识，可能 达到原子级别的分辨率。 了解生物钟的进展将对我们的 对行为和生理的调节，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，产生 睡眠-觉醒周期紊乱，代谢综合征，癌症发病率增加。那里 时钟功能障碍和情绪障碍之间也有联系，包括双相情感障碍。这个 拟议的研究旨在提供对生物的分子机制的见解 对健康至关重要的计时系统。

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.