THE PATHWAY THAT SETS THE CYANOBACTERIAL CIRCADIAN CLOCK

描述(摘自申请者摘要)：蓝藻是最简单的 已知的探索昼夜节律生物机制的生物 钟表。我们已经确定了一种名为cika的基因，它是转播不可或缺的。 用于重置生物钟的相位的环境信息 聚球藻原核生物昼夜节律模式系统--PCC7942 节奏研究。目的是阐明时钟的分子基础 以及它对光环境的夹带，我们将利用这一发现 通过确定Cika是否直接作为光感受器和通过识别 它的信号伙伴及其生化活动。Cika是以下组织的成员 光敏色素蛋白家族，但缺少预期的半胱氨酸残基 这为光敏色素中的胆琳发色团的结合提供了配体 其他一些类似光敏色素的蛋白质。C-终端结构域具有 保守的组氨酸蛋白激酶基序，并与 细菌反应调节蛋白的受体结构域。 我们将直接从蓝藻中纯化Cika，以确定是否有 发色团是附着的，如果是这样，它的化学特性和对Cika的影响 功能。这将通过修改Cika基因来增加一个 将其与蛋白质亲和标记，证实修饰后的基因是 通过对Cika零突变体的互补而发挥功能。的作用 重置时组氨酸蛋白激酶结构域的自动磷酸化将是 评估过了。通过酵母双杂交试验和转座子进行遗传筛选 在蓝藻中的诱变，将被用来识别与 Cika互动。这些Cika合作伙伴在阶段重置输入中的角色 路径也将被确定。除了提供基本的 对这一最基本的生物过程的洞察，该项目还 可能会发现目前尚不清楚的光敏色素的功能特性 存在于不同原核生物基因组中的同源物。

DESCRIPTION (Adapted from applicant's abstract): Cyanobacteria are the simplest organisms known in which to explore the mechanisms of circadian biological clocks. We have identified a gene, cikA, which is integral to relaying environmental information to reset the phase of the circadian clock in Synechococcus sp. strain PCC 7942, the model system for prokaryotic circadian rhythm studies. With the goal of elucidating the molecular basis of the clock and its entrainment to the photic environment, we will exploit this discovery by determining whether CikA acts directly as a photoreceptor and by identifying its signaling partners and their biochemical activities. CikA is a member of the phytochrome family of proteins, but lacks the expected cysteine residue that provides a ligand for bilin chromophore attachment in phytochromes and some other phytochrome-like proteins. The C-terminal domain has a well-conserved histidine protein kinase motif, and a segment of similarity with the receiver domains of response regulator proteins of bacteria. We will purify CikA directly from the cyanobacterium to determine whether a chromophore is attached, and if so, its chemical identity and influence on CikA function. This will be facilitated by modifying the cikA gene to add an affinity tag to the protein, and confirming that the modified gene is functional through complementation of a cikA null mutant. The role of autophosphorylation of the histidine protein kinase domain in resetting will be assessed. Genetic screens, both by a yeast two-hybrid assay and transposon mutagenesis in the cyanobacterium, will be used to identify proteins with which CikA interacts. The roles of these CikA partners in the phase resetting input pathway will be determined as well. In addition to providing fundamental insights into this most basic of biological processes, the project is also likely to uncover functional properties of currently cryptic phytochrome homologs that are present in the genomes of diverse prokaryotes.