kaiC is a gene belonging to the kaiABC gene cluster in several species of bacteria, including the cyanobacterium Synechococcus elongatus. Regulation of kaiABC expression is essential for circadian rhythmicity in Synechococcus. Using bacterial luciferase as a reporter for gene expression, studies have shown that, similar to Drosophila, mouse, and Neurospora clock models, the Synechococcus circadian clock is based on a negative feedback loop.
The name of this gene comes from the Japanese word kai, which translates to "cycle". Evolutionarily, kaiC is the oldest prokaryotic circadian gene with homologs that occur in Archaea and Proteobacteria. In contrast, kaiA is the most recent development and is thought to only have evolved in cyanobacteria. kaiC has a double-domain structure and sequence that classifies it as part of the RecA gene family of ATP-dependent recombinases. Based on a number of single-domain homologous genes in other species, kaiC is hypothesized to have horizontally transferred from Bacteria to Archaea, eventually forming the double-domain kaiC through duplication and fusion.
Genetics and protein structure
On S. elongatus's singular circular chromosome, the protein-coding gene kaiC is located at position 380696-382255 (its locus tag is syc0334_d). The gene kaiC has paralogs kaiB (located 380338..380646) and kaiA (located 379394..380248). kaiC encodes the protein KaiC (519 amino acids). KaiC acts as a promotor non-specific transcription repressor and its crystal structure has been solved at 2.8 Å resolution; it is a homohexameric complex (approximately 360 kDa) with a double-doughnut structure and a central pore which is open at the N-terminal ends and partially sealed at the C-terminal ends due to the presence of six arginine residues. The homo-hexamer has twelve ATP molecules between the N- and C-terminal domains. KaiC has two P loops or Walker’s motif As (ATP-/GTP-binding motifs) in the CI and CII domains; the CI domain also contains two DXXG (X represents any amino acid) motifs that are highly conserved among the GTPase super-family.
Cyanobacteria are the simplest organisms for which a mechanism is known for the generation of circadian rhythms. kaiA, kaiB, and kaiC have been shown to be essential genetic components in Synechococcus elongatus for circadian rhythms. Variations in the C-terminal region of each of their proteins suggest functional divergence between the Kai clock proteins, however there are critical interdependencies between the three paralogs. Protein KaiA enhances the phosphorylation of protein KaiC through control of the autophosphatase activity, while protein KaiB attenuates the activity of KaiA. Experiments have also shown that KaiC enhances the KaiA-KaiB interaction in yeast cells and in vitro. This implies that there may be the formation of a heteromultimeric complex composed of the three Kai proteins with KaiC serving as a bridge between KaiA and KaiB. Alternatively, KaiC may form a heterodimer with KaiA or KaiB to induce a conformational change.
Kai proteins regulate genome-wide gene expression. Disruption of the P loop in KaiC’s CI domain results both in arrhythmia and a reduction of ATP-binding activity; this, along with in vitro autophosphorylation of KaiC indicate that ATP binding to KaiC is crucial for Synechococcus circadian oscillation. The phosphorylation status of KaiC has been correlated with "Synechococcus" clock speed in vivo 
KaiC phosphorylation oscillates with a period of approximately 24 hours when placed in vitro with the three recombinant Kai proteins, incubated with ATP. The circadian rhythm of KaiC phosphorylation persists in constant darkness, regardless of Synechococcus transcription rates. This oscillation rate is thought to be controlled by the ratio of phosphorylated to unphosphorylated KaiC protein. KaiC phosphorylation ratio is a main factor in the activation of kaiBC promoter as well. The kaiBC operon is transcribed in a circadian fashion and precedes KaiC build up by about 6 hours, a lag thought to play a role in feedback loops. Scientists, such as Erin O'Shea, take advantage of this in vitro oscillator to provide a well-defined system in which to combine mathematics and experimentation in order to study the mechanism of a robust biological oscillator. KaiC is temperature compensated from 25 to 50 degrees Celsius  and has a Q10 of about 1.1. Because the KaiC period of oscillation is temperature compensated and agrees with in vivo circadian rhythms, KaiC is thought to be the mechanism for basic circadian timing in Synechococcus. ∆kaiABC individuals, one of the more common mutants, grow just as well as wild type individuals but lack rhythmicity. This is evidence that the kaiABC gene cluster is not necessary for growth.
Discovery and notable research
The relationship of kaiC to circadian oscillation in Synechoccus was first remarked upon in the 1998 paper “Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria” by Ishiura et al. Using a reporter to monitor the expression of clock-controlled gene psbAI in Synechoccus, Ishiura et al. investigated and reported on the rescue to normal rhythmicity of long-period clock mutant C44a by kaiABC.
A 2005 paper by Nakajima et al. details the first successful reconstruction of a circadian clock in vitro. Researchers incubated KaiA, KaiB, and KaiC in ratios similar to those in vivo in the presence of ATP in a test tube and observed that KaiC phosphorylation oscillated with a period close to 24 hours for several cycles without damping. The study also showed that the oscillation of KaiC phosphorylation has a Q10 of about 1.1, which is consistent with the in vivo, temperature compensated rhythm of gene expression. Additionally, the amount of in vitro KaiC remained constant, indicating that KaiC phosphorylation is both autonomous and sustainable. These features (temperature compensation, autonomous and sustainable oscillation, period of approximately 24 hours) are all characteristics of a circadian oscillator; the fact that they were exhibited by a system as simple as 3 proteins and ATP "in vitro" suggest the presence of an intriguing post-translational circadian mechanism.
The recreation of a circadian oscillation in vitro in the presence of only KaiA, KaiB, KaiC, and ATP has sparked interest in the relationship between cellular biochemical oscillators and their associated transcription,translation feedback loops (TTFLs). TTFLs have long been assumed to be the core of circadian rhythmicity, but that claim is now being tested against due to the possibility that the biochemical oscillators could constitute the central mechanism of the clock system, regulating and operating within TTFLs that control output and restore proteins essential to the oscillators in organisms such as Synechoccus, KaiABC.
A 2012 study out of Vanderbilt University shows evidence that KaiC acts as a phospho-transferase that hands back phosphates to ADP on the T432 (threonine residue at position 432) and S431 (serine residue 431) indicating that KaiC effectively serves as an ATP synthase.
- Ishiura, M. 1998. Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria. Science.
- Ishiura M, Kutsuna S, Aoki S, Iwasaki H, Andersson C R, Tanabe A, Golden S S, Johnson C H, Kondo T. (1998) Science 281:1519–1523
- Dvornyk, V. (2003) Origin and evolution of circadian clock genes in prokaryotes. PNAS 100:2495-2500
- Egli, M., Mori, T., Pattanayek, R., Xu, Y., Qin, X., & Johnson, C. H. (2012). Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism. Biochemistry, 51(8), 1547-1558.
- Nishiwaki T, Iwasaki H, Ishiura M, Kondo. 2000. Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria. Proc Natl Acad Sci U S A 97: 495–499.
- Murayama, Y., Oyama, T., & Kondo, T. (2008). Regulation of circadian clock gene expression by phosphorylation states of KaiC in cyanobacteria. Journal of bacteriology, 190(5), 1691-1698.
- Tomita, J., M. Nakajima, T. Kondo, and H. Iwasaki. 2005. No transcription-translation feedback in circadian rhythm of KaiC phosphorylation. Science 307:251-254.
- Xu, Y., T. Mori, and C. H. Johnson. 2003. Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC.EMBO J. 22:2117-2126
- Iwasaki, H., Taniguchi, Y., Ishiura, M., & Kondo, T. (1999). Physical interactions among circadian clock proteins KaiA, KaiB and KaiC in cyanobacteria. The EMBO journal, 18(5), 1137-1145.
- Xu, Y., Mori, T.,&Johnson, C. (2003). Cyanobacterial circadian clockwork: roles of KaiA, KaiB, and the "kaiBC" promoter in regulating KaiC. The EMBO Journal, 22, 2117-2126.
- Joseph S. Markson, Erin K. O’Shea (2009). The molecular clockwork of a protein-based circadian oscillator. FEBS Letters, 583(24), 3938-3947.
- Murakami, R., Miyake, A., Iwase, R., Hayashi, F., Uzumaki, T., & Ishiura, M. (2008). ATPase activity and its temperature compensation of the cyanobacterial clock protein KaiC. Genes to Cells, 13(4), 387-395.
- Terauchi, K., Y. Kitayama, T. Nishiwaki, K. Miwa, Y. Murayama, T. Oyama, and T. Kondo. 2007. ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria. Proc. Natl. Acad. Sci. USA104:16377-16381.
- Nakajima M, Imai K, Ito H, Nishiwaki T, Murayama Y, Iwasaki H, Oyama T, Kondo T. 2005. Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science 308:414-5.
- Egli M, Johnson CH. 2013. A circadian clock nanomachine that runs without transcription or translation. Curr. Opin. Neurobiol. http://dx.doi.org/10.1016/j.conb.2013.02.012 [Epub ahead of print]