Circadian Oscillators: Around the Transcription-Translation Feedback Loop and on to Output

Circadian clocks are ubiquitous timing systems that induce rhythms of biological activities in synchrony with night and day. In cyanobacteria, timing is generated by a posttranslational clock consisting of KaiA, KaiB, and KaiC proteins and a set of output signaling proteins, SasA and CikA, which transduce this rhythm to control gene expression. Here, we describe crystal and nuclear magnetic resonance structures of KaiB-KaiC,KaiA-KaiB-KaiC, and CikA-KaiB complexes. They reveal how the metamorphic properties of KaiB, a protein that adopts two distinct folds, and the post–adenosine triphosphate hydrolysis state of KaiC create a hub around which nighttime signaling events revolve, including inactivation of KaiA and reciprocal regulation of the mutually antagonistic signaling proteins, SasA and CikA.

/pdf/structural-basis-of-the-day-night-transition-in-a-bacterial-120uyfe7qo.pdf

Structural basis of the day-night transition in a bacterial circadian clock

The circadian nature of physiology and behavior is regulated by a circadian clock that generates intrinsic rhythms with a periodicity of approximately 24 h. The mammalian circadian system is composed of a hierarchical multi-oscillator structure, with the central clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus regulating the peripheral clocks found throughout the body. In the past two decades, key clock genes have been discovered in mammals and shown to be interlocked in transcriptional and translational feedback loops. At the cellular level, each cell is governed by its own independent clock; and yet, these cellular circadian clocks in the SCN form regional oscillators that are further coupled to one another to generate a single rhythm for the tissue. The oscillatory coupling within and between the regional oscillators appears to be critical for the extraordinary stability and the wide range of adaptability of the circadian clock, the mechanism of which is now being elucidated with newly advanced molecular tools.

/pdf/the-mammalian-circadian-system-a-hierarchical-multi-3ej292oy94.pdf

The mammalian circadian system: a hierarchical multi-oscillator structure for generating circadian rhythm.

Cyanobacteria have a robust circadian oscillator, known as the Kai system. Reconstituted from the purified protein components KaiC, KaiB, and KaiA, it can tick autonomously in the presence of adenosine 5'-triphosphate (ATP). The KaiC hexamers enter a natural 24-hour reaction cycle of autophosphorylation and assembly with KaiB and KaiA in numerous diverse forms. We describe the preparation of stoichiometrically well-defined assemblies of KaiCB and KaiCBA, as monitored by native mass spectrometry, allowing for a structural characterization by single-particle cryo-electron microscopy and mass spectrometry. Our data reveal details of the interactions between the Kai proteins and provide a structural basis to understand periodic assembly of the protein oscillator.

https://science.sciencemag.org/content/sci/355/6330/1181.full.pdf

Structures of the cyanobacterial circadian oscillator frozen in a fully assembled state

Chronobiological studies of prokaryotic organisms have generally lagged far behind the study of endogenous circadian clocks in eukaryotes, in which such systems are essentially ubiquitous. However, despite only being studied during the past 25 years, cyanobacteria have become important model organisms for the study of circadian rhythms and, presently, their timekeeping mechanism is the best understood of any system in terms of biochemistry, structural biology, biophysics and adaptive importance. Nevertheless, intrinsic daily rhythmicity among bacteria other than cyanobacteria is essentially unknown; some tantalizing information suggests widespread daily timekeeping among Eubacteria and Archaea through mechanisms that share common elements with the cyanobacterial clock but are distinct. Moreover, the recent surge of information about microbiome-host interactions has largely neglected the temporal dimension and yet daily cycles control important aspects of their relationship.

/pdf/timing-the-day-what-makes-bacterial-clocks-tick-4skjtdbyep.pdf

Timing the day: what makes bacterial clocks tick?

Circadian clocks generate slow and ordered cellular dynamics but consist of fast-moving bio-macromolecules; consequently, the origins of the overall slowness remain unclear. We identified the adenosine triphosphate (ATP) catalytic region [adenosine triphosphatase (ATPase)] in the amino-terminal half of the clock protein KaiC as the minimal pacemaker that controls the in vivo frequency of the cyanobacterial clock. Crystal structures of the ATPase revealed that the slowness of this ATPase arises from sequestration of a lytic water molecule in an unfavorable position and coupling of ATP hydrolysis to a peptide isomerization with high activation energy. The slow ATPase is coupled with another ATPase catalyzing autodephosphorylation in the carboxyl-terminal half of KaiC, yielding the circadian response frequency of intermolecular interactions with other clock-related proteins that influences the transcription and translation cycle.

https://science.sciencemag.org/content/sci/349/6245/312.full.pdf

Atomic-scale origins of slowness in the cyanobacterial circadian clock

KaiC, the core oscillator of the cyanobacterial circadian clock, is composed of an N-terminal C1 domain and a C-terminal C2 domain, and assembles into a double-ring hexamer upon ATP binding. Cyclic phosphorylation and dephosphorylation at Ser431 and Thr432 in the C2 domain proceed with a period of approximately 24 h in the presence of other clock proteins, KaiA and KaiB, but recent studies have revealed a crucial role for the C1 ring in determining the cycle period. In this study, we mapped dynamic structural changes of the C1 ring in solution using a combination of site-directed tryptophan mutagenesis and fluorescence spectroscopy. We found that the C1 ring undergoes a structural transition, coupled with ATPase activity and the phosphorylation state, while maintaining its hexameric ring structure. This transition triggered by ATP hydrolysis in the C1 ring in specific phosphorylation states is a necessary event for recruitment of KaiB, limiting the overall rate of slow complex formation. Our results provide structural and kinetic insights into the C1-ring rearrangements governing the slow dynamics of the cyanobacterial circadian clock.

/pdf/conformational-rearrangements-of-the-c1-ring-in-kaic-measure-12ehw7d9k6.pdf

Conformational rearrangements of the C1 ring in KaiC measure the timing of assembly with KaiB.

KaiC, a core protein of the cyanobacterial circadian clock, is rhythmically autophosphorylated and autodephosphorylated with a period of approximately 24 h in the presence of two other Kai proteins, KaiA and KaiB. In vitro experiments to investigate the KaiC phosphorylation cycle consume considerable time and effort. To automate the fractionation, quantification, and evaluation steps, we developed a suite consisting of an automated sampling device equipped with an 8-channel temperature controller and accompanying analysis software. Eight sample tables can be controlled independently at different temperatures within a fluctuation of ±0.01°C, enabling investigation of the temperature dependency of clock activities simultaneously in a single experiment. The suite includes an independent software that helps users intuitively conduct a densitometric analysis of gel images in a short time with improved reliability. Multiple lanes on a gel can be detected quasi-automatically through an auto-detection procedure implemented in the software, with or without correction for lane ‘smiling.’ To demonstrate the performance of the suite, robustness of the period against temperature variations was evaluated using 32 datasets of the KaiC phosphorylation cycle. By using the software, the time required for the analysis was reduced by approximately 65% relative to the conventional method, with reasonable reproducibility and quality. The suite is potentially applicable to other clock or clock-related systems in higher organisms, relieving users from having to repeat multiple manual sampling and analytical steps.

https://www.jstage.jst.go.jp/article/biophysico/13/0/13_235/_pdf

Jun Abe

Papers

Atomic-scale origins of slowness in the cyanobacterial circadian clock

Conformational rearrangements of the C1 ring in KaiC measure the timing of assembly with KaiB.

Accelerating in vitro studies on circadian clock systems using an automated sampling device.