Molecular mechanism of the Neurospora circadian clock

 描述（由申请人提供）：生物钟控制真核生物体中各种基本的细胞、生理和行为过程。允许测量时间的分子机制被称为“昼夜节律钟”，其输出为昼夜节律。我们的长期目标是了解生物钟的分子和生化机制。丝状真菌粗糙脉孢菌（Neurospora crassa）拥有最好的生物钟系统之一，为在分子水平上探索生物钟机制提供了一个强大的实验可用系统。与高等真核生物中的昼夜节律振荡器一样，脉孢菌振荡器由一个自动调节负反馈回路组成。在这个核心负反馈环中，两个白色蛋白（WC-1和WC-2）是形成WC复合物的正元件，该复合物激活频率（frq）基因的转录。负元件是FRQ和FRH，一种FRQ相互作用的RNA解旋酶，其形成抑制WCC活性的复合物。FRQ的转录和转录后调控在调控细胞凋亡中起着重要作用。 时钟在本提案中，我们旨在解决时钟机制的几个基本问题。 在具体目标1中，我们将确定FRQ的磷酸化如何调节其活性及其结构构象。本研究将有助于建立脉孢菌昼夜负反馈过程的关键生化机制。生物钟基因转录的节律性激活是真核生物生物钟的一个重要过程。我们最近发现CATP作为一个关键的时钟组件，控制frq转录影响染色质结构的frq基因座。在具体目标2中，我们将确定CATP如何通过调节染色质结构来调节frq转录的机制。反义RNA存在于脉孢菌属的frq位点和动物的per位点。我们最近证明，表达的frq反义RNA qrf是必不可少的时钟功能。在具体目标3中，我们 确定qrf转录如何通过转录干扰调节frq表达的机制。总之，这些目标利用了一个完善的模型系统，以解决三个基本问题，这是我们理解真核生物昼夜节律钟的关键，并将阐明脉孢菌时钟的遗传，生化和分子机制。由于脉孢菌和动物时钟之间的保守性，我们的研究结果将提供重要的见解真核生物的时钟机制。

 DESCRIPTION (provided by applicant): Circadian clocks control a wide variety of fundamental cellular, physiological, and behavioral processes in eukaryotic organisms. The molecular machinery that permits the measurement of time is referred to as the "circadian clock" and its output as circadian rhythms. Our long-term goal is to understand the molecular and biochemical mechanisms of circadian clocks. The filamentous fungus Neurospora crassa, which has one of the best understood circadian clock systems, offers a powerful experimentally-accessible system for exploring the clock mechanism at molecular levels. Like circadian oscillators in the higher eukaryotic organisms, the Neurospora oscillator consists of an autoregulatory negative feedback loop. In this core negative feedback loop, two WHITE COLLAR proteins (WC-1 and WC-2) are the positive elements that form a WC complex that activate the transcription of the frequency (frq) gene. The negative elements are FRQ and FRH, a FRQ- interacting RNA helicase, which form a complex that inhibits the WCC activity. The transcriptional and posttranscriptional regulation of FRQ plays central roles in the control of the clock. In this proposal, we aim to address several fundamental questions of the clock mechanism. In Specific Aim 1, we will determine how phosphorylation of FRQ regulates its activity and its structural conformation. This study will help establish a biochemical mechanism critical for the circadian negative feedback process in Neurospora. Rhythmic activation of clock gene transcription is an essential process in eukaryotic circadian clocks. We recently discovered CATP as a critical clock component that controls frq transcription by affecting chromatin structure at the frq locus. In Specific Aim 2, we will determine the mechanism for how CATP regulates frq transcription by regulating the chromatin structure. Antisense RNAs are present at the frq locus in Neurospora and per loci in animals. We recently demonstrated that the expression of the frq antisense RNA qrf is essential for clock function. In Specific Aim 3, we will determine the mechanism for how qrf transcription regulates frq expression by transcriptional interference. Together, these objectives take advantage of a well-established model system to address three fundamental questions that are critical for our understanding of eukaryotic circadian clocks and will elucidate the genetic, biochemical, and molecular mechanism of the Neurospora clock. Because of the conservation between the Neurospora and animal clocks, our results will provide important insights into eukaryotic clock mechanisms.