Kim Yeon Jeong, Somers David E
Department of Molecular Genetics, The Ohio State University, Columbus, OH, United States.
Front Plant Sci. 2019 May 22;10:667. doi: 10.3389/fpls.2019.00667. eCollection 2019.
Control of protein turnover is a key post-translational control point in the oscillatory network of the circadian clock. Some elements, such as TOC1 and PRR5 are engaged by a well-described F-box protein, ZEITLUPE, dedicated to their proteolytic turnover to shape their expression profile to a specific time of night. For most other clock components the regulation of their protein abundance is unknown, though turnover is often rapid and often lags the cycling of the respective mRNA. Here we report the design and results of an unbiased genetic screen in Arabidopsis to uncover proteolytic regulatory factors of PSEUDO-RESPONSE REGULATOR 7 (PRR7), a transcriptional repressor that peaks in the late afternoon. We performed EMS mutagenesis on a transgenic line expressing a () translational fusion that accurately recapitulates the diurnal and circadian oscillations of the endogenous PRR7 protein. Using continuous luciferase imaging under constant light, we recovered mutants that alter the PRR7-LUC waveform and some that change period. We have identified novel alleles of and , core components of the ELF3-ELF4-LUX Evening Complex (EC), that dampen the oscillation of PRR7-LUC. We report the characterization of two new hypomorphic alleles of that help to understand the relationship between molecular potency and phenotype.
蛋白质周转的控制是生物钟振荡网络中一个关键的翻译后控制点。一些元件,如TOC1和PRR5,会被一种描述详尽的F-box蛋白ZEITLUPE作用,该蛋白专门负责它们的蛋白水解周转,从而将它们的表达谱塑造为夜晚的特定时间。对于大多数其他生物钟组件而言,尽管它们的周转通常很快且常常滞后于各自mRNA的循环,但它们蛋白质丰度的调节机制尚不清楚。在此,我们报告了在拟南芥中进行的一项无偏向性遗传筛选的设计与结果,以揭示伪响应调节因子7(PRR7)的蛋白水解调节因子,PRR7是一种在下午晚些时候达到峰值的转录抑制因子。我们对一个表达()翻译融合体的转基因品系进行了EMS诱变,该融合体准确地重现了内源性PRR7蛋白的昼夜和生物钟振荡。在持续光照下使用连续荧光素酶成像,我们筛选到了改变PRR7-LUC波形的突变体以及一些改变周期的突变体。我们鉴定出了ELF3-ELF4-LUX夜间复合体(EC)的核心组件ELF3和ELF4的新等位基因,它们会减弱PRR7-LUC的振荡。我们报告了两个新的低表达等位基因的特征,这有助于理解分子效力与表型之间的关系。