Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America.
PLoS Biol. 2010 Jun 15;8(6):e1000394. doi: 10.1371/journal.pbio.1000394.
Cyanobacteria are the only model circadian clock system in which a circadian oscillator can be reconstituted in vitro. The underlying circadian mechanism appears to comprise two subcomponents: a post-translational oscillator (PTO) and a transcriptional/translational feedback loop (TTFL). The PTO and TTFL have been hypothesized to operate as dual oscillator systems in cyanobacteria. However, we find that they have a definite hierarchical interdependency-the PTO is the core pacemaker while the TTFL is a slave oscillator that quickly damps when the PTO stops. By analysis of overexpression experiments and mutant clock proteins, we find that the circadian system is dependent upon the PTO and that suppression of the PTO leads to damped TTFL-based oscillations whose temperature compensation is not stable under different metabolic conditions. Mathematical modeling indicates that the experimental data are compatible with a core PTO driving the TTFL; the combined PTO/TTFL system is resilient to noise. Moreover, the modeling indicates a mechanism by which the TTFL can feed into the PTO such that new synthesis of clock proteins can phase-shift or entrain the core PTO pacemaker. This prediction was experimentally tested and confirmed by entraining the in vivo circadian system with cycles of new clock protein synthesis that modulate the phosphorylation status of the clock proteins in the PTO. In cyanobacteria, the PTO is the self-sustained core pacemaker that can operate independently of the TTFL, but the TTFL damps when the phosphorylation status of the PTO is clamped. However, the TTFL can provide entraining input into the PTO. This study is the first to our knowledge to experimentally and theoretically investigate the dynamics of a circadian clock in which a PTO is coupled to a TTFL. These results have important implications for eukaryotic clock systems in that they can explain how a TTFL could appear to be a core circadian clockwork when in fact the true pacemaker is an embedded biochemical oscillator.
蓝藻是唯一能够在体外重建生物钟振荡器的模型昼夜节律钟系统。潜在的昼夜节律机制似乎包括两个亚组件：翻译后振荡器 (PTO) 和转录/翻译反馈环 (TTFL)。PTO 和 TTFL 被假设为在蓝藻中作为双振荡器系统运行。然而，我们发现它们具有明确的层次相互依存关系-PTO 是核心起搏器，而 TTFL 是当 PTO 停止时快速衰减的从属振荡器。通过对过表达实验和突变生物钟蛋白的分析，我们发现生物钟系统依赖于 PTO，并且抑制 PTO 会导致基于 TTFL 的衰减振荡，其温度补偿在不同代谢条件下不稳定。数学模型表明，实验数据与核心 PTO 驱动 TTFL 的情况相兼容；组合的 PTO/TFFL 系统对噪声具有弹性。此外，该模型表明了 TTFL 可以反馈到 PTO 的机制，从而使新的时钟蛋白合成可以相移或使核心 PTO 起搏器同步。该预测通过用新的时钟蛋白合成的循环来驯化体内昼夜节律系统进行了实验测试和证实，该循环调节 PTO 中时钟蛋白的磷酸化状态。在蓝藻中，PTO 是自我维持的核心起搏器，可以独立于 TTFL 运行，但是当 PTO 的磷酸化状态被钳制时，TTFL 会衰减。但是，TTFL 可以为 PTO 提供驯化输入。这项研究是首次在实验和理论上研究与 TTFL 耦合的 PTO 的昼夜节律钟动力学的研究。这些结果对于真核生物钟系统具有重要意义，因为它们可以解释当真正的起搏器是嵌入式生化振荡器时，TTFL 如何表现为核心生物钟。
