Figure 15–18C and D). In Drosophila and many other animals, including humans, the heart of the circadian clock is a delayed negative feedback loop based on transcription regulators: accumulation of certain gene products switches off the transcription of their own genes, but with a delay, so that the cell oscillates between a state in which the products are present and transcription is switched off, and one in which the products are absent and transcription is switched on (Figure 15–66). The negative feedback underlying circadian rhythms does not have to be based on transcription regulators. In some cell types, the circadian clock is constructed of proteins that govern their own activities through post-translational mechanisms, as we discuss next.
The best understood circadian clock is found in the photosynthetic cyanobacterium, Synechococcus elongatus. The core oscillator in this organism is remarkably simple, being composed of just three proteins—KaiA, KaiB, and KaiC. The central player is KaiC, a multifunctional enzyme that catalyzes its own phosphorylation and dephosphorylation in a 24-hour cycle: it gradually phosphorylates itself sequentially at two sites during the day and dephosphorylates itself during the night. This timing depends on interactions with the two other Kai proteins: KaiA binds to unphosphorylated KaiC and stimulates KaiC autophosphosphorylation, first at one site and then, with a delay, at the other. The second phosphorylation promotes the binding of the third protein, KaiB, which blocks the stimulatory effect of KaiA and thereby allows KaiC to dephosphorylate itself, bringing KaiC back to its dephosphorylated state. This clock depends on a negative feedback loop: KaiC drives its own phosphorylation until, after a delay, it recruits an inhibitor, KaiB, that stimulates KaiC to dephosphorylate itself. Amazingly, when the three Kai proteins are purified and incubated in a test tube with ATP, KaiC phosphorylation and dephosphorylation occur with roughly 24-hour timing over a period of several days (Figure 15–67).
Circadian oscillations in KaiC phosphorylation lead to parallel rhythms in the expression of large numbers of genes involved in controlling metabolic activities and cell division (see Figure 15–67). As a result, many aspects of cell behavior are synchronized with the circadian cycle.
Even in continuous darkness, cyanobacterial cells generate free-running oscillations of KaiC phosphorylation with roughly 24-hour periods. As in other circadian clocks, the cyanobacterial clock is entrained by the environmental light/dark
Figure 15–66 Simplified outline of the mechanism of the circadian clock in Drosophila cells. A central feature of the clock is the periodic accumulation and decay of two transcription regulatory proteins, Tim (short for timeless, based on the phenotype of a gene mutation) and Per (short for period). The mRNAs encoding these proteins rise gradually during the day and are translated in the cytosol, where the two proteins associate to form a heterodimer. After a time delay, the heterodimer dissociates and Tim and Per are transported into the nucleus, where Per represses the Tim and Per genes, resulting in negative feedback that causes the levels of Tim and Per to fall. In addition to this transcriptional feedback, the clock depends on numerous other proteins. For example, the controlled degradation of Per indicated in the diagram imposes delays in the accumulation of Tim and Per, which are crucial to the functioning of the clock. Steps at which specific delays are imposed are shown in red.