Post-translational Oscillator Research Articles

Insights into a clock's fidelity through vesicular encapsulation.

The single-celled cyanobacterium, Synechococcus elongatus , generates circadian rhythms with exceptional fidelity and synchrony despite their femtoliter volumes. Here, we explore the mechanistic aspects of this fidelity, by reconstituting the KaiABC post-translational oscillator (PTO) in cell-mimetic giant vesicles (GUVs) under well-defined conditions in vitro . PTO proteins were encapsulated with a coefficient of variation that closely matched protein variations observed in live cells. Using fluorescently labeled KaiB and confocal microscopy, we were able to measure circadian rhythms generated by thousands of encapsulated PTOs at the single-vesicle level for several days as a function of protein concentration and GUV size. We find that PTO fidelity decreased with decreasing levels of encapsulated PTO proteins and in smaller GUVs. We also observed that in encapsulated PTOs, a significant fraction of KaiB localized to GUV membranes like it does in cyanobacteria. A mathematical model that uses empirical bulk concentration and stoichiometry limitations suggests that cyanobacteria overcome challenges to fidelity by expressing high levels of PTO proteins along with the CikA and SasA proteins, which buffer stochastic variations in the levels of KaiA and KaiB, respectively. Further, the model suggests that the transcription-translation feedback loop (TTFL) contributes at most a small percentage to the overall fidelity of the cyanobacterial circadian clock under constant conditions but is essential for maintaining phase synchrony. Our results are the first experimental demonstration of populations of synthetic cells that can autonomously keep circadian time. Additionally, the approach of using bulk relationships to understand complex phenomena in cell-like systems could be useful for understanding other collective behavior important in biology, such as liquid-liquid phase separation.

A geometrically controlled post-translational oscillator for synthetic biology

A geometrically controlled post-translational oscillator for synthetic biology

Roles for the Synechococcus elongatus RNA-Binding Protein Rbp2 in Regulating the Circadian Clock.

The cyanobacterial circadian oscillator, consisting of KaiA, KaiB, and KaiC proteins, drives global rhythms of gene expression and compaction of the chromosome and regulates the timing of cell division and natural transformation. While the KaiABC posttranslational oscillator can be reconstituted in vitro, the Kai-based oscillator is subject to several layers of regulation in vivo. Specifically, the oscillator proteins undergo changes in their subcellular localization patterns, where KaiA and KaiC are diffuse throughout the cell during the day and localized as a focus at or near the pole of the cell at night. Here, we report that the CI domain of KaiC, when in a hexameric state, is sufficient to target KaiC to the pole. Moreover, increased ATPase activity of KaiC correlates with enhanced polar localization. We identified proteins associated with KaiC in either a localized or diffuse state. We found that loss of Rbp2, found to be associated with localized KaiC, results in decreased incidence of KaiC localization and long-period circadian phenotypes. Rbp2 is an RNA-binding protein, and it appears that RNA-binding activity of Rbp2 is required to execute clock functions. These findings uncover previously unrecognized roles for Rbp2 in regulating the circadian clock and suggest that the proper localization of KaiC is required for a fully functional clock in vivo.

Dynamic behavior of the cyanobacterial circadian clock with regulation of CikA* *Project supported by the National Natural Science Foundation of China (Grant No. 11672177).

Cyanobacteria are the simplest organisms to have circadian clocks. The central oscillator in cyanobacteria is composed by a transcriptional/translational feedback loop (TTFL) and a post-translational oscillator (PTO). The PTO is a core pacemaker which consists of three proteins KaiA, KaiB and KaiC. KaiA stimulates the phosphorylation of KaiC, while KaiB inhibits the activity of KaiA. The cyanobacterial circadian clock is an interesting topic for researchers and many mathematical models have been constructed. However, the current mathematical models of the cyanobacterial circadian clock have been made only considering the interactions between Kai proteins. CikA, as an input pathway component, plays an essential role in the circadian clock, whose mutation results in abnormal rhythms. The regulation mechanism of CikA remains unclear. In this paper, we develop a detailed mathematical model for the cyanobacterial circadian clock with incorporation CikA-regulation. Based on numerical simulations, we explore the dynamic properties of the circadian clock regulated by CikA. The results show that the regulation of CikA makes the system more sensitive. In detail, CikA strengthens the central role of PTO and improves the adaptability of the circadian clock against the change of environment. With CikA, the system is able to modulate its period more easily to face environmental perturbation. CikA also enhances slightly the fitness of cyanobacteria. The findings of this paper can supplement the biological research and may help us more clearly understand the cyanobacterial circadian clock regulated by other proteins.

Non-sinusoidal Waveform in Temperature-Compensated Circadian Oscillations

Time series of biological rhythms are of various shapes. Here, we investigated the waveforms of circadian rhythms in gene-protein dynamics using a newly developed, to our knowledge, index to quantify the degree of distortion from a sinusoidal waveform. In general, most biochemical reactions accelerate with increasing temperature, but the period of circadian rhythms remains relatively stable with temperature change, a phenomenon known as “temperature compensation.” Despite extensive research, the mechanism underlying this remains unclear. To understand the mechanism, we used transcriptional-translational oscillator models for circadian rhythms in the fruit fly Drosophila and mammals. Given the assumption that reaction rates increase with temperature, mathematical analyses revealed that temperature compensation required waveforms that are more nonsinusoidal at higher temperatures. We then analyzed a post-translational oscillator (PTO) model of cyanobacteria circadian rhythms. Because the structure of the PTO is different from that of the transcriptional-translational oscillator, the condition for temperature compensation would be expected to differ. Unexpectedly, the computational analysis again showed that temperature compensation in the PTO model required a more nonsinusoidal waveform at higher temperatures. This finding held for both models even with a milder assumption that some reaction rates do not change with temperature, which is consistent with experimental evidence. Together, our theoretical analyses predict that the waveform of circadian gene-activity and/or protein phosphorylation rhythms would be more nonsinusoidal at higher temperatures, even when there are differences in the network structures.

The Kai-Protein Clock-Keeping Track of Cyanobacteria's Daily Life.

Life has adapted to Earth'sday-night cycle with the evolution of endogenous biological clocks. Whereas these circadian rhythms typically involve extensive transcription-translation feedback in higher organisms, cyanobacteria have a circadian clock, which functions primarily as a protein-based post-translational oscillator. Known as the Kai system, it consists of three proteins KaiA, KaiB, and KaiC. In this chapter, we provide a detailed structural overview of the Kai components and how they interact to produce circadian rhythms of global gene expression in cyanobacterial cells. We discuss how the circadian oscillation is coupled to gene expression, intertwined with transcription-translation feedback mechanisms, and entrained by input from the environment. We discuss the use of mathematical models and summarize insights into the cyanobacterial circadian clock from theoretical studies. The molecular details of the Kai system are well documented for the model cyanobacterium Synechococcus elongatus, but many less understood varieties of the Kai system exist across the highly diverse phylum of Cyanobacteria. Several species contain multiple kai-gene copies, while others like marine Prochlorococcus strains have a reduced kaiBC-only system, lacking kaiA. We highlight recent findings on the genomic distribution of kai genes in Bacteria and Archaea and finally discuss hypotheses on the evolution of the Kai system.

High protein copy number is required to suppress stochasticity in the cyanobacterial circadian clock

Circadian clocks generate reliable ~24-h rhythms despite being based on stochastic biochemical reactions. The circadian clock in Synechococcus elongatus uses a post-translational oscillator that cycles deterministically in a test tube. Because the volume of a single bacterial cell is much smaller than a macroscopic reaction, we asked how clocks in single cells function reliably. Here, we show that S. elongatus cells must express many thousands of copies of Kai proteins to effectively suppress timing errors. Stochastic modeling shows that this requirement stems from noise amplification in the post-translational feedback loop that sustains oscillations. The much smaller cyanobacterium Prochlorococcus expresses only hundreds of Kai protein copies and has a simpler, hourglass-like Kai system. We show that this timer strategy can outperform a free-running clock if internal noise is significant. This conclusion has implications for clock evolution and synthetic oscillator design, and it suggests hourglass-like behavior may be widespread in microbes.

Roles for ClpXP in regulating the circadian clock in Synechococcus elongatus

In cyanobacteria, the KaiABC posttranslational oscillator drives circadian rhythms of gene expression and controls the timing of cell division. The Kai-based oscillator can be reconstituted in vitro, demonstrating that the clock can run without protein synthesis and degradation; however, protein degradation is known to be important for clock function in vivo. Here, we report that strains deficient in the ClpXP1P2 protease have, in addition to known long-period circadian rhythms, an exaggerated ability to synchronize with the external environment (reduced "jetlag") compared with WT strains. Deletion of the ClpX chaperone, but not the protease subunits ClpP1 or ClpP2, results in cell division defects in a manner that is dependent on the expression of a dusk-peaking factor. We propose that chaperone activities of ClpX are required to coordinate clock control of cell division whereas the protease activities of the ClpXP1P2 complex are required to maintain appropriate periodicity of the clock and its synchronization with the external environment.

Design Principles of Phosphorylation-Dependent Timekeeping in Eukaryotic Circadian Clocks.

The circadian clock in cyanobacteria employs a posttranslational oscillator composed of a sequential phosphorylation-dephosphorylation cycle of KaiC protein, in which the dynamics of protein structural changes driven by temperature-compensated KaiC's ATPase activity are critical for determining the period. On the other hand, circadian clocks in eukaryotes employ transcriptional feedback loops as a core mechanism. In this system, the dynamics of protein accumulation and degradation affect the circadian period. However, recent studies of eukaryotic circadian clocks reveal that the mechanism controlling the circadian period can be independent of the regulation of protein abundance. Instead, the circadian substrate is often phosphorylated at multiple sites at flexible protein regions to induce structural changes. The phosphorylation is catalyzed by kinases that induce sequential multisite phosphorylation such as casein kinase 1 (CK1) with temperature-compensated activity. We propose that the design principles of phosphorylation-dependent circadian-period determination in eukaryotes may share characteristics with the posttranslational oscillator in cyanobacteria.

Period Robustness and Entrainability of the Kai System to Changing Nucleotide Concentrations

Circadian clocks must be able to entrain to time-varying signals to keep their oscillations in phase with the day-night rhythm. On the other hand, they must also exhibit input compensation: their period must remain approximately one day in different constant environments. The posttranslational oscillator of the Kai system can be entrained by transient or oscillatory changes in the ATP fraction, yet is insensitive to constant changes in this fraction. We study in three different models of this system how these two seemingly conflicting criteria are met. We find that one of these (our recently published Paijmans model) exhibits the best tradeoff between input compensation and entrainability: on the footing of equal phase-response curves, it exhibits the strongest input compensation. Performing stochastic simulations at the level of individual hexamers allows us to identify a new, to our knowledge, mechanism, which is employed by the Paijmans model to achieve input compensation: at lower ATP fraction, the individual hexamers make a shorter cycle in the phosphorylation state space, which compensates for the slower pace at which they traverse the cycle.

Architecture and mechanism of the central gear in an ancient molecular timer

Molecular clocks are the product of natural selection in organisms from bacteria to human and their appearance early in evolution such as in the prokaryotic cyanobacterium Synechococcus elongatus suggests that these timers served a crucial role in genetic fitness. Thus, a clock allows cyanobacteria relying on photosynthesis and nitrogen fixation to temporally space the two processes and avoid exposure of nitrogenase carrying out fixation to high levels of oxygen produced during photosynthesis. Fascinating properties of molecular clocks are the long time constant, their precision and temperature compensation. Although these are hallmarks of all circadian oscillators, the actual cogs and gears that control clocks vary widely between organisms, indicating that circadian timers evolved convergently multiple times, owing to the selective pressure of an environment with a daily light/dark cycle. In S. elongatus, the three proteins KaiA, KaiB and KaiC in the presence of ATP constitute a so-called post-translational oscillator (PTO). The KaiABC PTO can be reconstituted in an Eppendorf tube and keeps time in a temperature-compensated manner. The ease by which the KaiABC clock can be studied in vitro has made it the best-investigated molecular clock system. Over the last decade, structures of all three Kai proteins and some of their complexes have emerged and mechanistic aspects have been analysed in considerable detail. This review focuses on the central gear of the S. elongatus clock and only enzyme among the three proteins: KaiC. Our determination of the three-dimensional structure of KaiC early in the quest for a better understanding of the inner workings of the cyanobacterial timer revealed its unusual architecture and conformational differences and unique features of the two RecA-like domains constituting KaiC. The structure also pinpointed phosphorylation sites and differential interactions with ATP molecules at subunit interfaces, and helped guide experiments to ferret out mechanistic aspects of the ATPase, auto-phosphorylation and auto-dephosphorylation reactions catalysed by the homo-hexamer. Comparisons between the structure of KaiC and those of nanomachines such as F1-ATPase and CaMKII also exposed shared architectural features (KaiC/ATPase), mechanistic principles (KaiC/CaMKII) and phenomena, such as subunit exchange between hexameric particles critical for function (clock synchronization, KaiABC; memory-storage, CaMKII).

Systems Biology-Derived Discoveries of Intrinsic Clocks.

A systems approach to studying biology uses a variety of mathematical, computational, and engineering tools to holistically understand and model properties of cells, tissues, and organisms. Building from early biochemical, genetic, and physiological studies, systems biology became established through the development of genome-wide methods, high-throughput procedures, modern computational processing power, and bioinformatics. Here, we highlight a variety of systems approaches to the study of biological rhythms that occur with a 24-h period—circadian rhythms. We review how systems methods have helped to elucidate complex behaviors of the circadian clock including temperature compensation, rhythmicity, and robustness. Finally, we explain the contribution of systems biology to the transcription–translation feedback loop and posttranslational oscillator models of circadian rhythms and describe new technologies and “–omics” approaches to understand circadian timekeeping and neurophysiology.

Protein-Protein Interactions in the Cyanobacterial Circadian Clock: Structure of KaiA Dimer in Complex with C-Terminal KaiC Peptides at 2.8 Å Resolution.

In the cyanobacterial circadian clock, the KaiA, -B, and -C proteins with ATP constitute a post-translational oscillator. KaiA stimulates the KaiC autokinase, and KaiB antagonizes KaiA action. KaiA contacts the intrinsically disordered C-terminal regions of KaiC hexamer to promote phosphorylation across subunit interfaces. The crystal structure of KaiA dimer from Synechococcus elongatus with two KaiC C-terminal 20mer peptides bound reveals that the latter adopt an α-helical conformation and contact KaiA α-helical bundles via mostly hydrophobic interactions. This complex and the crystal structure of KaiC hexamer with truncated C-terminal tails can be fit into the electron microscopy (EM) density of the KaiA:KaiC complex. The hybrid model helps rationalize clock phenotypes of KaiA and KaiC mutants.

Structural and biophysical methods to analyze clock function and mechanism.

Structural approaches have provided insight into the mechanisms of circadian clock oscillators. This review focuses upon the myriad structural methods that have been applied to the molecular architecture of cyanobacterial circadian proteins, their interactions with each other, and the mechanism of the KaiABC posttranslational oscillator. X-ray crystallography and solution NMR were deployed to gain an understanding of the three-dimensional structures of the three proteins KaiA, KaiB, and KaiC that make up the inner timer in cyanobacteria. A hybrid structural biology approach including crystallography, electron microscopy, and solution scattering has shed light on the shapes of binary and ternary Kai protein complexes. Structural studies of the cyanobacterial oscillator demonstrate both the strengths and the limitations of the divide-and-conquer strategy. Thus, investigations of complexes involving domains and/or peptides have afforded valuable information into Kai protein interactions. However, high-resolution structural data are still needed at the level of complexes between the 360-kDa KaiC hexamer that forms the heart of the clock and its KaiA and KaiB partners.

An arginine tetrad as mediator of input-dependent and input-independent ATPases in the clock protein KaiC.

A post-translational oscillator (PTO) composed of the proteins KaiA, KaiB and KaiC is at the heart of the cyanobacterial circadian clock. KaiC interacts with KaiA and KaiB over the daily cycle, and CII domains undergo rhythmic phosphorylation/dephosphorylation with a 24 h period. Both the N-terminal (CI) and C-terminal (CII) rings of KaiC exhibit ATPase activity. The CI ATPase proceeds in an input-independent fashion, but the CII ATPase is subject to metabolic input signals. The crystal structure of KaiC from Thermosynechococcus elongatus allows insight into the different anatomies of the CI and CII ATPases. Four consecutive arginines in CI (Arg linker) that connect the P-loop, CI subunits and CI and CII at the ring interface are primary candidates for the coordination of the CI and CII activities. The mutation of linker residues alters the period or triggers arhythmic behavior. Comparison between the CI and CII structures also reveals differences in loop regions that are key to KaiA and KaiB binding and activation of CII ATPase and kinase. Common packing features in KaiC crystals shed light on the KaiB-KaiC interaction.

Attenuation of the posttranslational oscillator via transcription–translation feedback enhances circadian-phase shifts in Synechococcus

Circadian rhythms are endogenous biological timing processes that are ubiquitous in organisms ranging from cyanobacteria to humans. In the photoautotrophic unicellular cyanobacterium Synechococcus elongatus PCC 7942, under continuous light (LL) conditions, the transcription-translation feedback loop (TTFL) of KaiC generates a rhythmic change in the accumulation of KaiC relative to KaiA clock proteins (KaiC/KaiA ratio), which peak and trough at subjective dawn and dusk, respectively. However, the role of TTFL in the cyanobacterial circadian system remains unclear because it is not an essential requirement for the basic oscillation driven by the Kai-based posttranslational oscillator (PTO) and the transcriptional output mechanisms. Here, we show that TTFL is important for the circadian photic resetting property in Synechococcus. The robustness of PTO, which is exemplified by the amplitude of the KaiC phosphorylation cycle, changed depending on the KaiC/KaiA ratio, which was cyclic under LL. After cells were transferred from LL to the dark, the clock protein levels remained constant in the dark. When cells were transferred from LL to continuous dark at subjective dawn, the KaiC phosphorylation cycle was attenuated with a lower KaiC/KaiA ratio, a higher KaiC phosphorylation level, and a lower amplitude than that in cells transferred at subjective dusk. We also found that the greater the degree to which PTO was attenuated in continuous dark, the greater the phase shifts upon the subsequent light exposure. Based on these results, we propose that TTFL enhances resetting of the Kai-based PTO in Synechococcus.

A Molecular Dynamics Study of the Cyanobacterial Clock Protein KaiA

Regulation of daily physiological functions with a ~24-hour periodicity, or circadian rhythms, exists in both eukaryotes and prokaryotes. So far, cyanobacteria are only known prokaryotes proved to have circadian rhythmicity. The circadian system in cyanobacteria comprises a post-translational oscillator (PTO) and a transcriptional/translational feedback loop (TTFL). The PTO comprise of three proteins (KaiA, KaiB, KaiC), and can be reconstituted in vitro with the existence of ATP. Phase of the PTO is associated with the phosphorylation states of KaiC, with KaiA promoting the phosphorylation of KaiC, and KaiB promoting the de-phosphorylation. Here we studied the dynamics of the KaiA protein ofThermosynechococcus elongatus. The result will be helpful in understanding the function of KaiA and its binding with KaiC.

CryoEM and Molecular Dynamics of the Circadian KaiB–KaiC Complex Indicates That KaiB Monomers Interact with KaiC and Block ATP Binding Clefts

The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. The KaiB–KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein–protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.

Analysis of the Sequence Conservation of the Circadian Clock Protein KaiC

Regulation of daily physiological functions with approximate a 24-hour periodicity, or circadian rhythms, is a characteristic of eukaryotes. So far, cyanobacteria are only known prokaryotes reported to possess circadian rhythmicity. The circadian system in cyanobacteria comprises both a post-translational oscillator (PTO) and a transcriptional/translational feedback loop (TTFL). The PTO can be reconstituted in vitro with three purified proteins (KaiA, KaiB, and KaiC) with the existence of ATP. Phase of the nanoclockwork has been associated with the phosphorylation states of KaiC, with KaiA promoting the phosphorylation of KaiC, and KaiB de-phosphorylating KaiC. Here we studied the sequence variation of 65 KaiC proteins in evolution, and determined some key residues in KaiC by analyzing the site variation rates of the protein sequences. These key residues could be used to study the key interactions of KaiC with KaiA and KaiB.

Revealing a Two-Loop Transcriptional Feedback Mechanism in the Cyanobacterial Circadian Clock

Molecular genetic studies in the circadian model organism Synechococcus have revealed that the KaiC protein, the central component of the circadian clock in cyanobacteria, is involved in activation and repression of its own gene transcription. During 24 hours, KaiC hexamers run through different phospho-states during daytime. So far, it has remained unclear which phospho-state of KaiC promotes kaiBC expression and which opposes transcriptional activation. We systematically analyzed various combinations of positive and negative transcriptional feedback regulation by introducing a combined TTFL/PTO model consisting of our previous post-translational oscillator that considers all four phospho-states of KaiC and a transcriptional/translational feedback loop. Only a particular two-loop feedback mechanism out of 32 we have extensively tested is able to reproduce existing experimental observations, including the effects of knockout or overexpression of kai genes. Here, threonine and double phosphorylated KaiC hexamers activate and unphosphorylated KaiC hexamers suppress kaiBC transcription. Our model simulations suggest that the peak expression ratio of the positive and the negative component of kaiBC expression is the main factor for how the different two-loop feedback models respond to removal or to overexpression of kai genes. We discuss parallels between our proposed TTFL/PTO model and two-loop feedback structures found in the mammalian clock.