The Inner Workings of an Ancient Biological Clock

Abstract

Circadian clocks evolved in diverse organisms as an adaptation to the daily swings in ambient light and temperature that derive from Earth’s rotation. These timing systems, based on intracellular molecular oscillations, synchronize organisms’ behavior and physiology with the 24-hour environmental rhythm. The cyanobacterial clock serves as a special model for understanding circadian rhythms because it can be fully reconstituted in vitro. This review summarizes recent advances that leverage new biochemical, biophysical, and mathematical approaches to shed light on the molecular mechanisms of cyanobacterial Kai proteins that support the clock, and their homologues in other bacteria. Many questions remain in circadian biology, and the tools developed for the Kai system will bring us closer to the answers.

Biological timekeeping in cyanobacteria

Cyanobacteria comprise a phylogenetically distinct lineage of photosynthetic microorganisms that live in almost all aquatic and terrestrial environments on Earth [1]. Their need for light energy to support growth shapes their physiology. Around one billion years ago, a circadian clock evolved that confers better adaptation to the diel cycle by allowing cyanobacteria to anticipate environmental changes on a 24-hour timescale [2]. A wealth of genetic tools enables use of the cyanobacterium Synechococcus elongatus as a model organism to understand how circadian clocks work at the molecular level. This model system has revealed amazing features of an ancient prokaryotic timing system that has engaged biologists, biochemists, and physicists for three decades. In this review, we summarize the current understanding of this fascinating molecular machine, featuring recent works that apply new experimental and theoretical methods.

The KaiABC circadian oscillator

Circadian rhythms in S. elongatus are built on a post-translational oscillator (PTO) that is reinforced by a transcription-translation feedback loop (TTFL) (see Glossary). The clock proteins KaiA, KaiB, and KaiC [3] form a PTO that undergoes an approximately 24 h cycle of KaiC phosphorylation and dephosphorylation to generate an internal representation of local time [4,5]. The temporal information that is biochemically encoded by the PTO is used in turn to control the rhythmic expression of kai genes, adding an additional layer of genetic control to timekeeping [6]. Remarkably, the PTO can be decoupled from the TTFL in S. elongatus and maintain robust and coherent rhythmicity [5,7–9]. In vivo, cells maintain rhythmic gene expression even when the kai genes are driven by an orthologous promoter that lacks elements for clock regulation [7,9]. In vitro, the ~24 h phosphorylation-dephosphorylation cycle of KaiC can be reconstituted by mixing only the Kai proteins, Mg²⁺ and ATP [5]. The molecular rhythm of this reconstituted PTO, termed the in vitro oscillator (IVO), fulfills the defining criteria of a circadian clock (Box 1).

BOX 1: Criteria for circadian rhythms.

By convention, a biological rhythm can be called a circadian rhythm when it meets the following criteria:

Endogenous free-running period:

This feature refers to the duration of the natural cycle of the rhythm in the absence of external cues. For a rhythm to be considered circadian, it must persist even in constant conditions, such as constant darkness or constant light, with a period of ~24 h. This value is known as the free-running period. The elimination of external cues rules out the possibility that the rhythms are driven by rhythmic daily environmental changes.

Entrainment:

External environmental cues, such as light and temperature, can synchronize or reset the phasing of the internal circadian rhythm. This process is called entrainment. An external cue that resets the rhythm is called the zeitgeber, or “time giver”. The ability of the human biological clock to adjust to local time after traveling across time zones is an example of entrainment. Entrainment ensures that the internal clocks stay aligned with the external environment.

Temperature compensation:

Thermodynamic rules govern that biochemical reactions run faster at higher temperature. However, the circadian clock should not run faster in the summer than in the winter, because the length of the day is still 24 h. The ability of circadian rhythms to maintain a similar period length over a range of physiological temperatures is called temperature compensation.

The PTO undergoes a number of oscillatory events at the biochemical level, with most driven by daily changes in KaiC phosphorylation [10,11]. The KaiC protein, a member of the P-loop ATPases, is composed of two ATPase domains: the N-terminal CI domain, and the C-terminal CII domain, which also has autokinase and autophosphatase activities [7,10,12,13]. The intrinsic ATPase activity of KaiC is very low compared with other P-loop ATPases (~15 ATP hydrolyzed per day by KaiC vs 10³ −10⁷ ATP per day by others) [10]. The CI and CII domains form two distinct rings within the hexameric structure of KaiC [14]. In the presence of KaiA and KaiB, two residues on the CII domain of KaiC, S431 and T432, undergo an orderly autophosphorylation cycle with a ~24 h period (S,T->S,pT->pS,pT->pS,T->S,T->…, in which pS or pT denotes S431 or T432 phosphorylation) [11,15–17] (Figure 1).

Figure 1. The cyanobacterial circadian clock.

The cyanobacterium S. elongatus PCC 7492 has a circadian clock based on the KaiABC core oscillator. KaiA stimulates phosphorylation of KaiC while KaiB acts antagonistically to dampen this process. The result of these interactions is a ~24-h cycle of phosphorylation (yellow circle) and dephosphorylation of KaiC. Kai protein complexes in different phosphorylation states activate two functionally opposed histidine kinases, SasA and CikA. Both of these proteins act on the same DNA-binding response regulator, RpaA, with SasA promoting phosphorylation of RpaA and CikA acting oppositely. The phosphorylation state of RpaA favors its DNA binding activity; thus, the rhythmic phosphorylation of KaiC leads to the oscillation gene expression via the rhythmic phosphorylation of a transcription factor.

Due to its ATPase activity, CII-associated ATP can be hydrolyzed to ADP, and the ADP remains bound, even in the presence of ATP [18]. KaiA stimulates the autokinase activity of KaiC by binding to a C-terminal segment known as the A-loop and promoting the exchange of ADP for ATP [19,20]. As phosphorylation progresses, the CII hexamer changes in stability: phosphorylation of T432 destabilizes the CII ring, further promoting the KaiA-KaiC interaction, while phosphorylation of S431 tightens the CII ring and makes the A-loops less accessible to KaiA [21,22]. The phosphorylation state of KaiC is also influenced by magnesium binding, which may regulate the conformation of the A-loop independently of KaiA [23]. The structural consequences of KaiC phosphorylation on A-loop conformation and accessibility have been proposed to help synchronize the noisy oscillations of individual hexamers to generate population-level oscillations [21,24,25].

Formation of the KaiBC complex upon phosphorylation of S431 (in pSpT or pST) marks the day-to-night transition (Figure 1). Stacking of the CI and CII rings upon phosphorylation of S431 increases the affinity of KaiB for KaiC by exposing its binding site on the B-loop of the CI domain [26]. The binding of KaiB to KaiC is regulated by two additional molecular events. First, KaiB requires the CI domain to be in its ADP-bound state to bind [26–28]. The temperature insensitive hydrolysis of ATP by CI appears to drive timing in the S. elongatus circadian oscillator because the ATPase activity of CI mutants correlates well with circadian period lengths observed in vitro and in vivo [10,29,30]. Second, the tetrameric KaiB ground state undergoes a rare fold switch to a monomeric form that takes on a thioredoxin-like fold that is required to bind KaiC (Figure 2A) [31]. KaiB monomers are recruited to the KaiC hexamer in a highly cooperative manner [32,33], resulting in formation of a KaiB ring on the KaiC CI domain that stably sequesters KaiA [25,34]. The sequestration of KaiA in an autoinhibitory conformation closes the negative feedback loop of the PTO, allowing KaiC to autodephosphorylate. As this happens, the KaiC rings unstack and the KaiABC complex disassembles, transitioning from night to day to start a new cycle of phosphorylation (Figure 1).

Figure 2. Fold-switching and structural mimicry by KaiB.

(A) Structures of the ground state KaiB tetramer (PDB: 4KSO) and the monomeric fold-switched active state (PDB: 5JYT).

(B) Secondary structure of the ground state (gsKaiB) and fold-switched (fsKaiB) monomers, with a box depicting the region that undergoes fold switching.

(C) Tertiary structure of the ground state (gsKaiB) and fold-switched (fsKaiB) monomers, colored as in panel B. The monomeric fsKaiB exhibits structural mimicry of the thioredoxin-like domain of SasA (PDB: 1T4Y), shown at the right.

While the general picture of how the PTO of S. elongatus generates circadian rhythms is now well established, fundamental steps within the cycle still lack detailed mechanisms. The following sections introduce recent progress revealing new molecular details and the development of new assays that deepen the mechanistic clarity with which we can understand the Kai system.

Allosteric changes linking KaiC CI and CII domains

The formation of the KaiBC complex at night is a key step in the negative feedback required to maintain oscillations. However, only recently have we begun to understand how phosphorylation of the CII domain allosterically regulates KaiB binding on the CI domain located ~70 Å away. Several groups have begun to address this question using high-resolution crystallography and cryo-EM studies on KaiC phosphomimetic mutants that lock KaiC into day or nighttime-like states coupled with biochemistry [35,36]. This work revealed that phosphorylation of S431 is crucial for the allosteric regulation of CI domains by inducing a conformational change in the CII-α9 helix near the phosphorylation site that propagates through the CII domain to the CI-CII interface, and then on to the CI nucleotide-binding pocket, as described below.

The dynamic nature of the KaiC CII-ring structure, revealed first by biochemical studies and NMR [21,37] and later by cryo-EM [21,35], is lost in tight crystal packing [21,35]. Under cryo-EM conditions, the CII ring is destabilized in the daytime KaiC mimetic, whereas the nighttime KaiC mimetic has a rigid CII ring (Figure 3), consistent with solution biochemical data [21,37]. Single-particle analysis by cryo-EM of nighttime KaiC revealed two populations: one with C6-symmetry that has six molecules of ATP bound in the CII hexamer, and one with C2-symmetry that has ADP bound in two opposing CII subunits of the hexamer and ATP at the other subunits [35]. The ADP-bound subunits in the C2-symmetric complex adopt a compressed conformation, whereas the ATP-bound subunits are extended like the subunits in the C6-symmetric complex (Figure 3). The single-turn CII-α9 helix where S431 and T432 are located is lengthened when ADP is bound, suggesting how this switch may be used to initiate interdomain communication in KaiC; lengthening of the CII-α9 helix shifts the nearby CII-α8 helix toward the CI-CII interface [35]. Substitution of tyrosine 402 (Y402) on this helix can tune the period from 15 h to 6.6 days [30], demonstrating that changes at the CI-CII interface have powerful control over oscillator timing. An arginine tetrad in the CI domain [38] holds the protomers together via electrostatic interactions and bridges the conformational change from the CII-α8 helix directly into the nucleotide binding site in CI to influence the hydrolysis of ATP needed for KaiB binding. Mutation of residues involved in this long-distance allosteric communication eliminate the preferential affinity of KaiB for nighttime KaiC and disrupt circadian rhythms [35].

Figure 3. Day-night differences in the structure of KaiC.

(A) Structural models of daytime and nighttime KaiC revealed by cryo-EM, with subunits in shades of yellow or blue, respectively. Daytime KaiC has a rigid CI ring and a destabilized CII ring that prevents the reconstruction of its structure unless stabilized by fos-choline (PDB: 7S67). Nighttime KaiC adopts two populations: one with C6 symmetry that is similar to the stabilized daytime structure (PDB: 7S66), and one with C2 symmetry marked by compression of the CI and CII rings (PDB:7S65).

(B) Comparison of the extended and compressed protomers within the C2-symmetric nighttime KaiC. In the compressed protomers, phosphorylation of S431 induces a local conformational change in the CII-α9 helix and shifts the nearby CII-α8 helix toward the CI-CII interface, causing CI-CII domain stacking; ATP (white), ADP (green).

Communicating temporal information from the oscillator

The molecular-level oscillations of Kai protein activity and interactions translate into rhythmic biological processes via a short output pathway in S. elongatus. SasA, a histidine kinase, and CikA, a dual-function histidine kinase/phosphatase, have opposing effects on the response regulator RpaA that controls the transcription of downstream target genes [39]. Engagement with the Kai complex triggers SasA to promote the phosphorylation of RpaA at dusk and causes CikA to catalyze RpaA dephosphorylation later in the evening. The DNA binding of RpaA is increased whenever its receiver domain is phosphorylated, which leads to the activation of Class 1 genes and repression of Class 2 genes [37]. Conversely, the dephosphorylation of RpaA leads to the reduced expression of Class 1 genes and de-repression of Class 2 genes [40]. When not bound to KaiC, SasA has very low kinase activity toward RpaA, whereas free CikA acts as an efficient kinase for RpaA [39].

SasA binds preferentially to KaiC when S431 is phosphorylated, on the same site as KaiB, although it binds with much higher apparent affinity [21,28]. The N-terminal domain of SasA stably adopts the thioredoxin-like fold that is required to bind the KaiC B-loop [41], whereas KaiB needs to undergo a rare fold-switching process from its highly stable ground-state to an unstable metamorphic state to bind the B-loop of KaiC (Figure 2) [31]. Moreover, SasA is a dimer, so its apparent affinity for KaiC is significantly enhanced by avidity [42]. In contrast to SasA, CikA binds to KaiB when it is bound to the KaiC CI ring, competing with KaiA for the same binding site on KaiB [28,43]. With SasA and CikA relaying timing information, the oscillation of Kai protein complexes drives rhythmic phosphorylation of RpaA, leading to rhythmic gene expression.

Monitoring in vitro oscillations in high throughput

Monitoring circadian oscillations by separating the different phospho-states of KaiC on a protein gel, although intuitive for biochemists, is labor intensive and of low throughput, even with automation in sample collection and data analysis [44]. The LiWang lab developed a high-throughput assay that monitors changes in fluorescence anisotropy, detecting size-dependent changes in the tumbling of fluorescently-labeled KaiB that report on the formation and disassembly of large clock complexes in real time in a format that can be easily scaled down to a 384-well plate (Figure 4) [45].

Figure 4. A high-throughput assay allows reconstitution of an in vitro clock.

Using fluorescence anisotropy as read out, in vitro reactions containing the core oscillator and the output components can be monitored continuously in a plate reader. Each component in the IVC reaction (other than KaiC) can be labeled with fluorophore and monitored in an individual well in a microtiter plate.

A reaction matrix of massively parallel clock reactions set up with different concentrations of the Kai clock proteins allowed for a deep exploration of the biochemical parameters of the oscillator [42]. Stable oscillations happen only within a relatively small range of concentrations, although the addition of SasA or CikA at physiological levels can rescue reactions that fail due to insufficient amounts of KaiB or KaiA, respectively. This remarkable finding is based on a combination of competitive and cooperative interactions. Specifically, SasA rescues weak oscillators with substoichiometric KaiB through heterotropic positive cooperativity—binding of the SasA thioredoxin-like domain to KaiC stimulates KaiB binding at neighboring CI domains, enhancing the apparent affinity between KaiB and KaiC [42]. CikA competes with KaiA for the KaiBC complex and increases the concentration of free KaiA to stimulate KaiC phosphorylation [28,43], helping to compensate for insufficient KaiA [42,46].

Expanding from a minimal oscillator to include clock input/output

By including fluorescent reporters on the output kinases CikA or SasA, or the circadian transcription factor RpaA in parallel reactions, it is now possible to monitor the rhythmic binding of RpaA to a DNA segment from the kaiBC promoter in vitro as RpaA becomes rhythmically regulated by the kinases [42,47] (Figure 4). This “complete-clock” reconstitution reaction is termed an in vitro clock (IVC) to distinguish it from the KaiABC-only IVO that lacks the output pathway (i.e., the signal relay to transcription). The IVC can be driven by either CikA or SasA, just as strains of S. elongatus lacking either sasA or cikA can still keep rhythmic gene expression in vivo [48,49]. When the IVC is driven solely by CikA, RpaA is phosphorylated by free CikA during the day and dephosphorylated when CikA is bound to KaiBC complex during the night [39] (Figure 1, day-night). By contrast, the SasA-driven IVC relies on the phosphorylation of RpaA by SasA when it is bound to KaiC at dusk and RpaA’s slow intrinsic autodephosphorylation activity to generate the rhythm [39] (Figure 1, dusk-night).

Resetting the phase of the clock

The IVC was also used to study how the clock responds to phase-resetting cues like changes in the cellular ATP/ADP ratio, which oscillate over a 24-h light/dark cycle [50]. A cikA (Circadian Input Kinase A) deletion strain fails to reset the phase of gene expression rhythms in vivo in response to a dark pulse that lowers the ATP/ADP ratio [49,51]; however, the reconstituted KaiABC oscillator resets well in response to an ADP pulse in vitro, indicating that something else must explain in vivo resetting phenotype. The use of parallel IVC reactions with different clock components led to the observation that the inclusion of CikA increases the magnitude of ADP-induced phase-resetting by KaiABC, while SasA suppresses it [50]. The inclusion of both proteins counterbalances these opposing effects to reach an intermediate phase-resetting response similar to that found in vivo. The successful reconstitution of the cikA-deletion phenotype in vitro is a great example of the utility of the IVC to probe mechanisms of circadian timekeeping in cyanobacteria, including the unexpected finding that the kinases are not only parts of the input/output system, but also fundamental to the timekeeping function of the oscillator itself.

Detailed molecular mechanisms revealed by new modeling

Circadian biologist Arnold Eskin proposed a linear clock paradigm where input components receive environmental cues that synchronize a core oscillator to control rhythmic activities via discrete output components [52]. Previously, it was thought that the cyanobacterial circadian clock follows this paradigm of distinct input (CikA), timekeeping (KaiABC), and output (SasA/CikA) components. However, recent data reveal a stark incompatibility with this simple paradigm because the oscillator components directly sense environmental cues to play a role in input. More specifically, KaiA binds oxidized quinones and KaiC responds directly to decreased ATP/ADP ratios, both of which signal the onset of darkness to reset the circadian rhythm, [51,53–55]. Conversely, the output kinases SasA and CikA are directly involved in maintaining timekeeping (an oscillator role) [42], as well as regulating how the oscillator responds to environmental cues (an input role) [49,50,56].

Mathematical modeling plays an important role in helping to understand the nonlinear dynamics of stable oscillations. For the Kai protein oscillator, novel models are typically proposed based on kinetic data to help identify key steps or factors that are otherwise hard to determine by experimental methods [11,27,57–59].

Recently, the use of electron paramagnetic resonance (EPR) spectroscopy was coupled with modeling to study the formation of the KaiBC complex, identifying two subpopulations of KaiB, “neighborless” and “neighbored”, on the KaiC CI ring [60,61]. These two states allowed the modeling of cooperative binding of KaiB to KaiC, supporting a model proposed by Koda and Saito where binding enhancement is achieved by keeping the same KaiB-KaiC on-rate but lowering the off-rate when KaiB interacts with its neighboring KaiB on the CI ring [62]. The same may be true for positive heterotropic cooperativity with SasA [42]. The Koda and Saito model also suggests that the slowness of KaiBC-complex formation comes from a slow transition of KaiC to a binding-competent post-ATP hydrolysis state in CI, rather than the KaiB fold-switching process [62].

The KaiBC complex that sequesters KaiA during the night ensures the autodephosphorylation of KaiC, and it disassembles at dawn to free KaiA up to activate KaiC autophosphorylation during the day [28,34]. Bayesian modeling on KaiA-activated KaiC phosphorylation kinetics showed that a complete sequestration of KaiA by KaiBC is not necessary to prevent KaiC phosphorylation at night due to the ultrasensitive response of KaiC phosphorylation to KaiA concentrations [63]. In other words, KaiBC complexes need only to sequester KaiA below a certain threshold to stop KaiC phosphorylation. Moreover, recent work showed that KaiB binding to KaiC alone is enough to induce KaiC autodephosphorylation, further lowering the need to fully sequester KaiA [64]. Conversely, to start a new cycle, the KaiA released from KaiABC complexes must reach a threshold to induce KaiC phosphorylation. This ultrasensitivity, important to synchronize oscillations and maintain a circadian period under various ATP/ADP ratios, is proposed to rely on the differential affinity of KaiA for different nucleotide-bound states of KaiC in the CII domain [63].

The disassembly of Kai protein complexes has been studied far less than their formation. However, a recent report showed that the disassembly rate of the KaiBC complex is dependent on the CI ATPase activity and is temperature compensated, i.e., its disassembly rate remains constant over a range of physiological temperatures [65]. Furthermore, KaiA actively promotes the disassembly process [61,66]. The addition of KaiA to a preformed KaiBC complex leads to the fast formation of some KaiABC complex, followed by a gradual disassembly of both KaiABC and KaiBC complexes [61,66]. The ability of KaiA to disrupt the KaiBC complex relies on its binding to the KaiC A-loops in their exposed state, which, unlike buried A-loops, do not form a network of interactions that stabilize the CII ring [67]. Thus, KaiA-A loop interactions enhance the looseness of the CII ring and likely oppose the CI-CII ring stacking needed for KaiB-KaiC interactions.

Most current models center on the kinetics and thermodynamics of just the three Kai proteins. Because both CikA and SasA interact with Kai complexes to directly affect the oscillator, its output, and its synchronization with external cues [42,50], future models will be more powerful if they take into account these kinases and other factors. For example, mathematical modeling suggests that the newly discovered KaiB-interacting protein KidA functions as a recycling factor for fold-switched KaiB, enhancing the interaction of this rare and unstable active state with KaiC [68]. Although the inclusion of more players will inevitably increase the complexity of modeling, additional key steps in this complicated biological process could be potentially explained.

Mechanistic insights from distant KaiBC homologs

Potential homologs of Kai proteins are encoded by genomes outside of the cyanobacterial lineage [69]. Indeed, a KaiBC-based system may underlie a different type of biological timing mechanism found in other photosynthetic bacteria. These KaiA-less clocks are thought to work as hourglass timers instead of self-sustaining clocks, meaning that they need a daily change in environmental cues to set their 24-h cycle [70]. Although it has been long known that there is rhythmic gene expression in R. sphaeroides and it possesses homologs of S. elongatus KaiB and KaiC [71], its timing mechanism has not been studied in detail until recently.

The KaiBC system from R. sphaeroides was characterized using biochemical and structural methods to show that it likely functions as an hourglass timer [72]. Some notable differences from S. elongatus KaiB and KaiC underlie this change in mechanism. First, KaiC_Rs (KaiC from R. sphaeroides) has an extended C-terminal domain that forms a coiled-coil, bringing two KaiC hexamers together to form a surprising dodecamer in solution. KaiC_Rs has an exposed A-loop helix similar to that found in the KaiA-activated KaiC_Se (KaiC from S. elongatus) [67,73], presumably leading to its higher kinase activity and increased nucleotide exchange rate. Second, the ATPase activity of KaiC_RS is ~10-fold higher than KaiC_Se and it is not temperature compensated. Finally, KaiB_Rs also binds to the CI ring of the phosphomimetic mutant KaiC_Rs-S413E/S414E using a thioredoxin-like fold; however, this binding is not cooperative [67].

With the addition of KaiB_Rs under a nighttime-like low ATP/ADP ratio, KaiC_Rs autodephosphorylates, and the return to a daytime-like high ATP/ADP ratio brings KaiC_Rs back to its phosphorylated state [72]. This change in ATP/ADP ratio is physiologically relevant because R. sphaeroides increases its ATP/ADP ratio under light through photosynthesis, with lower ATP/ADP ratios in the dark [74]. Therefore, unlike KaiABC_Se, which maintains stable oscillations of phosphorylation over a range of ATP/ADP ratios [51], a feature termed metabolic compensation [75], KaiBC_Rs relies on changes in cellular ATP/ADP ratios to determine its phosphorylation status like an hourglass timer.

Although the KaiBC_Rs system does not comprise a self-sustaining circadian clock in vitro, we believe it could be coupled with a TTFL in vivo to achieve sustained oscillation. Notably, a kaiA deletion strain of S. elongatus can still oscillate, although it is severely damped, relying on rhythmic formation of the KaiBC complex and the TTFL [76]. By applying an external temperature cycle with a period ~24 h, the amplitude of the damped oscillation could be further enhanced via resonance [76]. A similar mechanism involving resonance of the TTFL with external light and/or temperature cycles could also be used by organisms with a KaiA-less timing mechanism.

Concluding remarks

Despite significant advancements in recent years, one of the most elusive mechanistic questions in circadian biology still remains—what are the molecular events that give rise to temperature compensation, the feature that allows this protein-based oscillator to behave as a reliable clock rather than a thermometer? The CI ATPase activity of KaiC is proposed to be a central feature of this insensitivity to temperature, although an enzyme that maintains a relatively constant rate of activity over a 10-15°C range of temperature challenges basic concepts of biochemistry. Thus, understanding how this insensitivity to temperature is achieved should shed light on a defining, yet enigmatic, feature of circadian rhythms. Other components in the cyanobacterial oscillator, such as KaiA and KaiB, could also contribute to temperature compensation, and the application of advanced biochemical and biophysical methods should continue to address these questions and generate mechanistic data for years to come. Drawing upon the study of biological timekeeping mechanisms from different organisms offers a chance to explore how they may have evolved from simple hourglass timers to self-sustaining circadian clocks. Building a comprehensive framework to understand ATP hydrolysis and phosphorylation in temperature-compensated KaiC_Se and temperature-sensitive KaiC_Rs, including a potential regulatory role for magnesium, could help provide an answer to this question from an evolutionary point of view in prokaryotes.

Glossary

Avidity

refers to the combined strength of multiple affinities of individual non-covalent binding interactions.

Fold switch

proteins are a subset of globular proteins whose stably formed structures change dramatically due to alternate folding pathways or in response to signaling or environmental cues.

Metabolic compensation

refers to homeostatic mechanisms that limit perturbation of timekeeping by circadian oscillators in response to changing metabolic factors in the cellular milieu.

Post-translational oscillator

describes a negative feedback loop that is independent of transcription or translation, usually mediated by a combination of post-translational modifications, conformational changes, and/or protein-protein interactions to generate a biochemical oscillation.

Transcription-translation feedback loops

generate rhythmic gene expression by negative feedback, wherein the product of a gene negatively regulates its own expression.