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.
Significance The Kai system is a widely studied model in theoretical biology and systems biology. It is to date the only known circadian clock that can be reconstituted in vitro. Essential to the rhythmicity of the system is the formation of the KaiC–KaiB complex. Many aspects of this interaction, such as the mode of binding of KaiB, the stoichiometry of the interaction, and the exact binding interfaces have long remained ambiguous. We present a mass spectrometry-based structural model of the KaiC–KaiB interaction that answers many of these outstanding questions on the basis of direct experimental evidence. This structural model sheds light on the intricate workings of the in vitro oscillator.
BackgroundCircadian clocks are found in organisms of almost all domains including photosynthetic Cyanobacteria, whereby large diversity exists within the protein components involved. In the model cyanobacterium Synechococcus elongatus PCC 7942 circadian rhythms are driven by a unique KaiABC protein clock, which is embedded in a network of input and output factors. Homologous proteins to the KaiABC clock have been observed in Bacteria and Archaea, where evidence for circadian behavior in these domains is accumulating. However, interaction and function of non-cyanobacterial Kai-proteins as well as homologous input and output components remain mainly unclear.ResultsUsing a universal BLAST analyses, we identified putative KaiC-based timing systems in organisms outside as well as variations within Cyanobacteria. A systematic analyses of publicly available microarray data elucidated interesting variations in circadian gene expression between different cyanobacterial strains, which might be correlated to the diversity of genome encoded clock components. Based on statistical analyses of co-occurrences of the clock components homologous to Synechococcus elongatus PCC 7942, we propose putative networks of reduced and fully functional clock systems. Further, we studied KaiC sequence conservation to determine functionally important regions of diverged KaiC homologs. Biochemical characterization of exemplary cyanobacterial KaiC proteins as well as homologs from two thermophilic Archaea demonstrated that kinase activity is always present. However, a KaiA-mediated phosphorylation is only detectable in KaiC1 orthologs.ConclusionOur analysis of 11,264 genomes clearly demonstrates that components of the Synechococcus elongatus PCC 7942 circadian clock are present in Bacteria and Archaea. However, all components are less abundant in other organisms than Cyanobacteria and KaiA, Pex, LdpA, and CdpA are only present in the latter. Thus, only reduced KaiBC-based or even simpler, solely KaiC-based timing systems might exist outside of the cyanobacterial phylum, which might be capable of driving diurnal oscillations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-017-0999-7) contains supplementary material, which is available to authorized users.
The coordination of biological activities into daily cycles provides an important advantage for the fitness of diverse organisms. Most eukaryotes possess an internal clock ticking with a periodicity of about one day to anticipate sunrise and sunset. The 24-hour period of the free-running rhythm is highly robust against many changes in the natural environment. Among prokaryotes, only Cyanobacteria are known to harbor such a circadian clock. Its core oscillator consists of just three proteins, KaiA, KaiB, and KaiC that produce 24-hour oscillations of KaiC phosphorylation, even in vitro. This unique three-protein oscillator is well documented for the freshwater cyanobacterium Synechococcus elongatus PCC 7942. Several physiological studies demonstrate a circadian clock also for other Cyanobacteria including marine species. Genes for the core clock components are present in nearly all marine cyanobacterial species, though there are large differences in the specific composition of these genes. In the first section of this review we summarize data on the model circadian clock from S. elongatus PCC 7942 and compare it to the reduced clock system of the marine cyanobacterium Prochlorococcus marinus MED4. In the second part we discuss the diversity of timing mechanisms in other marine Cyanobacteria with regard to the presence or absence of different components of the clock.
Highlights d TOP1 promotes RNAPII transcription and clearance from chromosomes in prometaphase d TOP1 assists RNAPII promoter loading during mitotic exit to restart transcription d Loss of TOP1-RNAPII interaction causes supercoil buildup and segregation defects d Disrupting TOP1-RNAPII binding affects growth and sensitizes cells to mTOR drugs
21Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation 22 strategies and differences in the presence of circadian clock proteins. In Synechococcus elongatus 23 PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity 24 of KaiC correlates with the period of the clock and mediates temperature compensation. 25 Synechocystis sp. PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins 26 were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore 27 characterized the enzymatic activity of KaiC3 as a representative of non-standard KaiC homologs 28 in vitro. KaiC3 displayed ATPase activity, which were lower compared to the Synechococcus 29 elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature-dependent. Hence, KaiC3 is 30 missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid 31 analysis showed that KaiC3 interacts with KaiB3, KaiC1 and KaiB1. Further, KaiB3 and KaiB1 32reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in 33 constant darkness is completely abolished after deletion of ∆kaiAB1C1 and reduced in the 34 absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a 35 crosstalk between the KaiC1 and KaiC3 based systems. 36 Importance: The circadian clock influences the cyanobacterial metabolism and deeper 37 understanding of its regulation will be important for metabolic optimizations in context of 38 industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock 39 systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is 40 required. Synechocystis PCC 6803 represents a major cyanobacterial model organism and harbors 41 phylogenetically diverged homologs of the clock proteins, which are present in various other non-42 3 cyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple 43 Synechocystis Kai proteins and characterize enzymatic activities of the non-standard clock 44 homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness 45 suggesting its involvement in the regulation of non-photosynthetic metabolic pathways. 46 Introduction: 47 Cyanobacteria have evolved the circadian clock system to sense, anticipate and respond to 48 predictable environmental changes based on the rotation of Earth and the resulting day-night 49 cycle. Circadian rhythms are defined by three criteria: (i) oscillations with a period of about 24 50 hours without external stimuli, (ii) synchronization of the oscillator with the environment and (iii) 51 compensation of the usual temperature dependence of biochemical reactions, so that the period 52 of the endogenous oscillation does not depend on temperature in a physiological range (2). The 53 cyanobacterial circadian clock system has been studied in much detail in the unicellular model 54 cyanoba...
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