Department of Biology, Kansai Medical University, Hirakata, Osaka, Japan.
J Bacteriol. 2013 Oct;195(19):4517-26. doi: 10.1128/JB.00300-13. Epub 2013 Aug 2.
In the cyanobacterium Synechococcus elongatus PCC7942, KaiA, KaiB, and KaiC are essential elements of the circadian clock, and Kai-based oscillation is thought to be the basic circadian timing mechanism. The Kai-based oscillator coupled with transcription/translation feedback and other intercellular factors maintains the stability of the 24-hour period in vivo. In this study, we showed that disruption of the Clp protease family genes clpP1, clpP2, and clpX and the overexpression of clpP3 cause long-period phenotypes. There were no significant changes in the levels of the clock proteins in these mutants. The overexpression of clpX led to a decrease in kaiBC promoter activity, the disruption of the circadian rhythm, and eventually cell death. However, after the transient overexpression of clpX, the kaiBC gene expression rhythm recovered after a few days. The rhythm phase after recovery was almost the same as the phase before clpX overexpression. These results suggest that the core Kai-based oscillation was not affected by clpX overexpression. Moreover, we showed that the overexpression of clpX sequentially upregulated ribosomal protein subunit mRNA levels, followed by upregulation of other genes, including the clock genes. Additionally, we found that the disruption of clpX decreased the expression of the ribosomal protein subunits. Finally, we showed that the circadian period was prolonged following the addition of a translation inhibitor at a low concentration. These results suggest that translational efficiency affects the circadian period and that clpX participates in the control of translation efficiency by regulating the transcription of ribosomal protein genes.
在蓝藻集胞藻 PCC7942 中，KaiA、KaiB 和 KaiC 是生物钟的必需元件，基于 Kai 的振荡被认为是基本的生物钟计时机制。基于 Kai 的振荡器与转录/翻译反馈和其他细胞间因素相结合，维持了体内 24 小时周期的稳定性。在这项研究中，我们表明 Clp 蛋白酶家族基因 clpP1、clpP2 和 clpX 的破坏以及 clpP3 的过表达会导致长周期表型。这些突变体中时钟蛋白的水平没有明显变化。clpX 的过表达导致 kaiBC 启动子活性降低，生物钟节律破坏，最终导致细胞死亡。然而，在 clpX 过表达后的短暂时间内，kaiBC 基因表达节律在几天后恢复。恢复后的节律相位几乎与 clpX 过表达前的相位相同。这些结果表明，核心基于 Kai 的振荡不受 clpX 过表达的影响。此外，我们表明 clpX 的过表达依次上调核糖体蛋白亚基 mRNA 水平，随后上调包括时钟基因在内的其他基因。此外，我们发现 clpX 的破坏降低了核糖体蛋白亚基的表达。最后，我们表明，在低浓度添加翻译抑制剂后，生物钟周期延长。这些结果表明翻译效率会影响生物钟周期，并且 clpX 通过调节核糖体蛋白基因的转录参与控制翻译效率。
