蓝藻生物钟分子机理的研究进展

蓝藻是具有内源性生物钟的简单生物.虽然蓝藻生物钟具有跟真核生物同样的基础特征,但其相关基因和蛋白质与真核生物没有同源性.蓝藻生物钟的核心是kai基因簇及其编码的蛋白KaiA,KaiB和KaiC.这三种Kai蛋白相互作用调节KaiC的磷酸化状态,从而产生昼夜节律信息.KaiC的磷酸化循环是昼夜节律的起博器,调控包括kai基因在内的相关基因的节律性表达.组氨酸蛋白激酶的磷酸化传递可将环境信息输入和将节律信息输出生物钟核心.     

The molecular clockwork of a protein-based circadian oscillator

The circadian clock of the cyanobacterium Synechococcuselongatus PCC 7942 is governed by a core oscillator consisting of the proteins KaiA, KaiB, and KaiC. Remarkably, circadian oscillations in the phosphorylation state of KaiC can be reconstituted in a test tube by mixing the three Kai proteins and adenosine triphosphate. The in vitro oscillator provides a well-defined system in which experiments can be combined with mathematical analysis to understand the mechanism of a highly robust biological oscillator. In this Review, we summarize the biochemistry of the Kai proteins and examine models that have been proposed to explain how oscillations emerge from the properties of the oscillator’s constituents.     

Biochemical Evidence for a Timing Mechanism in Prochlorococcus

Organisms coordinate biological activities into daily cycles using an internal circadian clock. The circadian oscillator proteins KaiA, KaiB, and KaiC are widely believed to underlie 24-h oscillations of gene expression in cyanobacteria. However, a group of very abundant cyanobacteria, namely, marine Prochlorococcus species, lost the third oscillator component, KaiA, during evolution. We demonstrate here that the remaining Kai proteins fulfill their known biochemical functions, although KaiC is hyperphosphorylated by default in this system. These data provide biochemical support for the observed evolutionary reduction of the clock locus in Prochlorococcus and are consistent with a model in which a mechanism that is less robust than the well-characterized KaiABC protein clock of Synechococcus is sufficient for biological timing in the very stable environment that Prochlorococcus inhabits.Cyanobacteria are photosynthetic prokaryotes that are known to possess a true circadian clock. Gene expression and other biological activities follow rhythmic cycles with a circa 24-h period. Rhythmic behavior is maintained even in the absence of environmental stimuli such as light and temperature. The underlying core oscillator consisting of the clock proteins KaiA, KaiB, and KaiC is the only characterized prokaryotic circadian oscillator. It was previously demonstrated that these three proteins, together with ATP, can produce 24-h oscillations of KaiC phosphorylation in vitro (17). The essential roles of KaiA and KaiB in oppositely influencing KaiC phosphorylation are well documented for the oscillator of “Synechococcus elongatus” PCC 7942 (hereafter S. elongatus), the species for which most bacterial circadian research has been conducted. Thus, it is puzzling that marine cyanobacteria of the genus Prochlorococcus, probably the most abundant photosynthetic organisms on Earth (5, 28), contain homologs of only two of these clock proteins, KaiC and KaiB (3, 11, 20). Laboratory cultures (10) as well as natural Prochlorococcus populations (24) display a rhythmic cell cycle together with a daily periodicity of gene expression that can be explained by the functioning of a circadian clock. Alternatively, these rhythms could be controlled directly by the daylight (10). The functional role of the Kai proteins from Prochlorococcus has remained entirely unclear and has not been experimentally addressed thus far.In the well-studied protein clock of S. elongatus, KaiC hexamers are at the center of the circadian oscillator, combining three intrinsic enzymatic activities: autokinase, autophosphatase, and ATPase. KaiA and KaiB modulate KaiC''s activities in opposite manners. KaiA seems to be essential for the shift between autophosphatase and autokinase, and for generating KaiC phosphorylation rhythms, by stabilizing C-terminal residues of KaiC, the A-loops (12). Thus, the absence of KaiA should have consequences for the enzymatic activities of the remaining Kai proteins of Prochlorococcus. In this study, the previously unknown functions of the Prochlorococcus sp. strain MED4 protein KaiB (ProKaiB) and ProKaiC are examined. In our in vitro experiments, we analyzed the recombinant proteins ProKaiB and ProKaiC in direct comparison to the core oscillator of S. elongatus, which consists of S. elongatus KaiA (SynKaiA), SynKaiB, and SynKaiC. We show here that both clock proteins from Prochlorococcus sp. strain MED4 independently exhibit their known biochemical functions, although the influence of ProKaiB on ProKaiC dephosphorylation is different certainly due to the absence of KaiA, the third protein of the oscillator. For ProKaiC, we demonstrate ATPase activity as well as the phosphorylation of serine 427 (S427) and threonine 428 (T428) using mass spectrometry and high-resolution sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Moreover, we suggest that the deletion of kaiA is compensated by the enhanced autophosphorylation activity of ProKaiC. Our results might have further implications for the analysis of a possible timing mechanism in other bacterial species, such as purple bacteria that encode KaiB and KaiC homologs but that lack the KaiA component.     

Timekeeping in bacteria: the cyanobacterial circadian clock

The prokaryotes known as cyanobacteria possess an endogenous 24h biological (circadian) clock that provides temporal coordination for physiological processes. Although the cyanobacterial clock has the same fundamental properties as circadian clocks in eukaryotes, its components are non-homologous to those of animals, plants or fungi. Moreover, its mechanism is likely to be very different from that depicted in eukaryotic clock models. The picture that is emerging for the timing mechanism in cyanobacteria is of a multiprotein, multimeric, molecular machine composed of proteins whose domains exhibit twists on common themes. Signal transduction into and out of the clock core appears to occur via histidine protein kinase-based phosphorylation relays.     

生物钟的转录后与翻译后水平调控进展

哺乳动物中的昼夜节律系统由位于下丘脑SCN核内的生物钟主钟和位于多数外周细胞中的子钟组成。在分子水平上,生物钟的节律振荡由生物钟基因及其编码蛋白的转录和翻译形成的自主的反馈环路组成,并接受外界因素的影响与环境周期保持同步。为此,就生物钟的调控机制而言,除了转录水平的基因表达调控外,生物钟转录产物和蛋白质的修饰也可以显著影响生物钟基因的表达时相。讨论了一些转录后与翻译后的修饰作用及其对生物钟的影响,并对其今后的研究方向作了展望。