Attenuation of the posttranslational oscillator via transcription-translation feedback enhances circadian-phase shifts in Synechococcus.
ABSTRACT: 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.
Project description:The circadian clock in the cyanobacterium Synechococcus elongatus is composed of a post-translational oscillator (PTO) that can be reconstituted in vitro from three different proteins in the presence of ATP and a transcription-translation feedback loop (TTFL). The homo-hexameric KaiC kinase, phosphatase and ATPase alternates between hypo- and hyper-phosphorylated states over the 24-h cycle, with KaiA enhancing phosphorylation, and KaiB antagonizing KaiA and promoting KaiC subunit exchange. SasA is a His kinase that relays output signals from the PTO formed by the three Kai proteins to the TTFL. Although the crystal structures for all three Kai proteins are known, atomic resolution structures of Kai and Kai/SasA protein complexes have remained elusive. Here, we present models of the KaiAC and KaiBC complexes derived from solution small angle X-ray scattering (SAXS), which are consistent with previous EM based models. We also present a combined SAXS/EM model of the KaiC/SasA complex, which has two N-terminal SasA sensory domains occupying positions on the C-terminal KaiC ring reminiscent of the orientations adopted by KaiB dimers. Using EM we demonstrate that KaiB and SasA compete for similar binding sites on KaiC. We also propose an EM based model of the ternary KaiABC complex that is consistent with the sequestering of KaiA by KaiB on KaiC during the PTO dephosphorylation phase. This work provides the first 3D-catalogue of protein-protein interactions in the KaiABC PTO and the output pathway mediated by SasA.
Project description:Cyanobacteria are unique organisms with remarkably stable circadian oscillations. These are controlled by a network architecture that comprises two regulatory factors: posttranslational oscillation (PTO) and a transcription/translation feedback loop (TTFL). The clock proteins KaiA, KaiB, and KaiC are essential for the circadian rhythm of the unicellular species Synechococcus elongatus PCC 7942. Temperature-compensated autonomous cycling of KaiC phosphorylation has been proposed as the primary oscillator mechanism that maintains the circadian clock, even in the dark, and it controls genome-wide gene expression rhythms under continuous-light conditions (LL). However, the kaiC(EE) mutation (where "EE" represents the amino acid changes Ser431Glu and Thr432Glu), where phosphorylation cycling does not occur in vivo, has a damped but clear kaiBC expression rhythm with a long period. This suggests that there must be coupling between the robust PTO and the "slave" unstable TTFL. Here, we found that the kaiC(EE) mutant strain in LL was hypersensitive to the dark acclimation required for phase shifting. Twenty-three percent of the genes in the kaiC(EE) mutant strain exhibited genome-wide transcriptional rhythms with a period of 48 h in LL. The circadian phase distribution was also conserved significantly in most of the wild-type and kaiC(EE) mutant strain cycling genes, which suggests that the output mechanism was not damaged severely even in the absence of KaiC phosphorylation cycles. These results strongly suggest that the KaiC phosphorylation cycle is not essential for generating the genome-wide rhythm under light conditions, whereas it is important for appropriate circadian timing in the light and dark.
Project description: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.
Project description:The kai gene cluster, which is composed of three genes, kaiA, kaiB and kaiC, is essential for the generation of circadian rhythms in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. Here we demonstrate the direct association of KaiA, KaiB and KaiC in yeast cells using the two-hybrid system, in vitro and in cyanobacterial cells. KaiC enhanced KaiA-KaiB interaction in vitro and in yeast cells, suggesting that the three Kai proteins were able to form a heteromultimeric complex. We also found that a long period mutation kaiA1 dramatically enhanced KaiA-KaiB interaction in vitro. Thus, direct protein-protein association among the Kai proteins may be a critical process in the generation of circadian rhythms in cyanobacteria.
Project description:In the unicellular cyanobacterium Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock under continuous light (LL) conditions. Here, we used high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild-type, kaiABC-null, and kaiC-overexpressor strains under LL and continuous dark (DD) conditions. In the wild-type strains, >30% of transcripts oscillated significantly in a circadian fashion, peaking at subjective dawn and dusk. Such circadian control was severely attenuated in kaiABC-null strains. Although it has been proposed that KaiC globally represses gene expression, our analysis revealed that dawn-expressed genes were up-regulated by kaiC-overexpression so that the clock was arrested at subjective dawn. Transfer of cells to DD conditions from LL immediately suppressed expression of most of the genes, while the clock kept even time in the absence of transcriptional feedback. Thus, the Synechococcus genome seems to be primarily regulated by light/dark cycles and is dramatically modified by the protein-based circadian oscillator.
Project description:Circadian rhythms are a fundamental property of most organisms, from cyanobacteria to humans. In the unicellular obligately photoautotrophic cyanobacterium Synechococcus elongatus PCC 7942, essentially all promoter activities are controlled by the KaiABC-based clock under continuous light conditions. When Synechococcus cells are transferred from the light to continuous dark (DD) conditions, the expression of most genes, including the clock genes kaiA and kaiBC, is rapidly down-regulated, whereas the KaiC phosphorylation cycle persists. Therefore, we speculated that the posttranslational oscillator might not drive the transcriptional circadian output without de novo expression of the kai genes. Here we show that the cyanobacterial clock regulates the transcriptional output even in the dark. The expression of a subset of genes in the genomes of cells grown in the dark was dramatically affected by kaiABC nullification, and the magnitude of dark induction was dependent on the time at which the cells were transferred from the light to the dark. Moreover, under DD conditions, the expression of some dark-induced gene transcripts exhibited temperature-compensated damped oscillations, which were nullified in kaiABC-null strains and were affected by a kaiC period mutation. These results indicate that the Kai protein-based posttranslational oscillator can drive the circadian transcriptional output even without the de novo expression of the clock genes.
Project description:The circadian clock of the cyanobacterium Synechococcus elongatus 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.
Project description: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).
Project description:The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by the KaiA, KaiB and KaiC proteins in the presence of ATP. The principal clock component, KaiC, undergoes regular cycles between hyper- and hypo-phosphorylated states with a period of ca. 24 h that is temperature compensated. KaiA enhances KaiC phosphorylation and this enhancement is antagonized by KaiB. Throughout the cycle Kai proteins interact in a dynamic manner to form complexes of different composition. We present a three-dimensional model of the S. elongatus KaiB-KaiC complex based on X-ray crystallography, negative-stain and cryo-electron microscopy, native gel electrophoresis and modelling techniques. We provide experimental evidence that KaiB dimers interact with KaiC from the same side as KaiA and for a conformational rearrangement of the C-terminal regions of KaiC subunits. The enlarged central channel and thus KaiC subunit separation in the C-terminal ring of the hexamer is consistent with KaiC subunit exchange during the dephosphorylation phase. The proposed binding mode of KaiB explains the observation of simultaneous binding of KaiA and KaiB to KaiC, and provides insight into the mechanism of KaiB's antagonism of KaiA.
Project description:In the unicellular cyanobacterium, Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock in continuous light (LL) conditions. Here, we employed high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild type, kaiABC-null and kaiC-overexpressor strains under LL and continuous dark (DD) conditions. In the wild type strain more than 30% of transcripts significantly oscillated in a circadian fashion, peaking at subjective dawn and dusk. Such circadian control was nullified in kaiABC-null strains. Although KaiC has been proposed to globally repress gene expression, our analysis revealed that dawn expressing genes were upregulated by kaiC-overexpression, such that the clock was arrested at subjective dawn. Transfer of cells to continuous dark (DD) conditions from LL immediately suppressed expression of most of genes, while the clock keeps time even in the absence of transcriptional feedback. Thus, the Synechococcus genome seems primarily regulated by the light/dark cycles and dramatically modified by the protein-based circadian oscillator. Keywords: timecourse data (~48 hours under continuous light or darkness) from Synechococcus elongatus PCC 7942 (wild type, kaiABC-null, and inducible kaiC-overexpressor) strains Overall design: Wild type (WT), kaiABC-null (Dkai), and inducible kaiC-overexpressor (oxC) S. elongatus PCC 7942 strains were analyzed under continuous light (LL), continuous dark (DD) using Affymetrix high-density oligonucleotide microarrays (GeneChip CustomExpress Arrays) representing predicted 2,515 protein-coding genes on the genome of Synechococcus elongatus PCC 6301, which can be used also to the almost homologous strain, S. elongatus PCC 7942: Two independent experiments in WT under LL (hours 0 to 52) and DD (hours 0 to 48); Two independent experiments in Dkai under LL (hours 0 to 48); Two independent experiments in oxC under LL in the presence or absence of an inducer, IPTG, from hour 25 to 33 in LL; a single experiment in WT under DD in the presence of an transcriptional inhibitor, rifampicin.