KaiC

KaiC is a gene belonging to the KaiABC gene cluster (with KaiA, and KaiB) that, together, regulate bacterial circadian rhythms, specifically in cyanobacteria. KaiC encodes for the KaiC protein which interacts with the KaiA and KaiB proteins in a post-translational oscillator (PTO). The PTO is cyanobacteria master clock that is controlled by sequences of phosphorylation of KaiC protein. Regulation of KaiABC expression and KaiABC phosphorylation is essential for cyanobacteria circadian rhythmicity, and is particularly important for regulating cyanobacteria processes such as nitrogen fixation, photosynthesis, and cell division. Studies have shown similarities to Drosophila, Neurospora, and mammalian clock models in that the kaiABC regulation of the cyanobacteria slave circadian clock is also based on a transcription translation feedback loop (TTFL). KaiC protein has both auto-kinase and auto-phosphatase activity and acts as the circadian regulator in both the PTO and the TTFL. KaiC has been found to not only suppress kaiBC when overexpressed, but also suppress circadian expression of all genes in the cyanobacterial genome. KaiC is a gene belonging to the KaiABC gene cluster (with KaiA, and KaiB) that, together, regulate bacterial circadian rhythms, specifically in cyanobacteria. KaiC encodes for the KaiC protein which interacts with the KaiA and KaiB proteins in a post-translational oscillator (PTO). The PTO is cyanobacteria master clock that is controlled by sequences of phosphorylation of KaiC protein. Regulation of KaiABC expression and KaiABC phosphorylation is essential for cyanobacteria circadian rhythmicity, and is particularly important for regulating cyanobacteria processes such as nitrogen fixation, photosynthesis, and cell division. Studies have shown similarities to Drosophila, Neurospora, and mammalian clock models in that the kaiABC regulation of the cyanobacteria slave circadian clock is also based on a transcription translation feedback loop (TTFL). KaiC protein has both auto-kinase and auto-phosphatase activity and acts as the circadian regulator in both the PTO and the TTFL. KaiC has been found to not only suppress kaiBC when overexpressed, but also suppress circadian expression of all genes in the cyanobacterial genome. Though the KaiABC gene cluster has been found to exist only in cyanobacteria, evolutionarily KaiC contains homologs that occur in Archaea and Proteobacteria. It is the oldest circadian gene that has been discovered in prokaryotes. 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. KaiC's key role in circadian control and homology to RecA suggest its individual evolution before its presence in the KaiABC gene cluster. Takao Kondo, Susan S. Golden, and Carl H. Johnson discovered the gene cluster in 1998 and named the gene cluster kaiABC, as 'kai' means “cycle” in Japanese. They generated 19 different clock mutants that were mapped to kaiA, kaiB, and kaiC genes, and successfully cloned the gene cluster in the cyanobacteria Synechococcus elongatus. Using a bacterial luciferase reporter to monitor the expression of clock-controlled gene psbAI in Synechococcus, they investigated and reported on the rescue to normal rhythmicity of long-period clock mutant C44a (with a period of 44 hours) by kaiABC. They inserted wild-type DNA through a pNIBB7942 plasmid vector into the C44a mutant, and generated clones that restored normal period (a period of 25 hours). They were eventually able to localize the gene region causing this rescue, and observed circadian rhythmicity in upstream promotor activity of kaiA and kaiB, as well as in the expression of kaiA and kaiBC messenger RNA. They determined abolishing any of the three kai genes would cause arrhythmicity in the circadian clock and reduce kaiBC promoter activity. KaiC was later found to have both autokinase and autophosphatase activity. These findings suggested that circadian rhythm was controlled by a TTFL mechanism, which is consistent with other known biological clocks. In 2000, S. elongatus was observed in constant dark (DD) and constant light (LL). In DD, transcription and translation halted due to the absence of light but the circadian mechanism showed no significant phase shift after transitioning to constant light. In 2005, after closer examination of the KaiABC protein interactions, the phosphorylation of KaiC proved to oscillate with daily rhythms in the absence of light. In addition to the TTFL model, the PTO model was hypothesized for the KaiABC phosphorylation cycle. Also in 2005, Nakajima et al. lysed S. elongatus and isolated KaiABC proteins. In test tubes containing only KaiABC proteins and ATP, in vitro phosphorylation of KaiC oscillated with a near 24 hour period with a slightly smaller amplitude than in vivo oscillation, proving that the KaiABC proteins are sufficient for circadian rhythm solely in the presence of ATP. Combined with the TTFL model, KaiABC as a circadian PTO was shown to be the fundamental clock regulator in S. elongatus On Synechococcus elongatus' 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 non-specific transcription regulator that represses transcription of the kaiBC promoter. 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 hexamer has twelve ATP molecules between the N- (CI) and C-terminal (CII) domains, which demonstrate ATPase activity. The CI and CII domains are linked by the N-terminal region of the CII domain. The last 20 residues from the C-terminal of the CII domain protrude from the doughnut to form what is called the A-loop. Interfaces on KaiC's CII domain are sites for both auto-kinase and auto-phosphatase activity, both in vitro and in vivo. 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. KaiC shares structural similarities to several other proteins with hexameric rings, including RecA, DnaB and ATPases.The hexameric rings of KaiC closely resembles RecA, with 8 α-helices surrounding a twisted β-sheet made up of 7 strands. This structure favours the binding of a nucleotide at the carboxy-end of the β-sheet. KaiC’s structural similarities to these proteins suggests a role for KaiC in transcription regulation. Further, the diameter of the rings in KaiC are suitable to accommodate single stranded DNA. Additionally, the surface potential at the CII ring and the C-terminal channel opening is mostly positive. The compatibility of the diameter as well as the surface potential charge suggests that DNA may be able to bind to the C-terminal channel opening. Kai proteins regulate genome-wide gene expression. Protein KaiA enhances the phosphorylation of protein KaiC by binding to the A loop of the CII domain to promote auto-kinase activity during subjective day. Phosphorylation at subunits occurs in an ordered manner, beginning with phosphorylation of Threonine 432 (T432) followed by Serine 431 (S431) on the CII domain. This leads to tight stacking of the CII domain to the CI domain. KaiB then binds to the exposed B loop on the CII domain of KaiC and sequesters KaiA from the C-terminals during subjective night, which inhibits phosphorylation and stimulates auto-phosphatase activity. Dephosphorylation of T432 occurs followed by S431, returning KaiC to its original state. Disruption of KaiC’s CI domain results both in arrhythmia of kaiBC expression 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. Additionally, overexpression of KaiC has been shown to strongly repress the kaiBC promoter, while kaiA overexpression has experimentally enhanced the kaiBC promoter. These positive and negative binding elements mirror a feedback mechanism of rhythm generation conserved across many different species.