User:Mominsiddiqui/sandbox

In molecular biology, the cyanobacterial clock proteins are the main circadian regulator in cyanobacteria. The proteins that comprise this circadian oscillator are KaiA, KaiB, and KaiC.

Discovery and Experimental Use
Due to the lack of a nucleus in these organisms and the fact that their cell cycle is shorter than 24 hours, there was doubt as to whether or not cyanobacteria would be able to express circadian rhythms. Huang et al. were the first to demonstrate that cyanobacteria do in fact have circadian rhythms when they discovered that the levels of nitrogen fixation in the Synechococcus bacteria oscillated with a light-entrainable 24-hour rhythm.

In a 1993 experiment, Kondo et al. used a luciferase reporter inserted into the genetically tractable Synechococcus, which was grown in a 12:12 light-dark cycle to ensure entrainment”.The two groups of bacteria were entrained in antiphase to each other so that while one group was experiencing 12 hours of light, the other was experiencing 12 hours of darkness. Once the bacteria entered the stationary phase, they were transferred into test tubes kept in constant light, except for 5-minute recording periods every 30 minutes, in which the tubes were kept in darkness to measure their levels of bioluminescence. They found that the level of bioluminescence cycled at a near 24-hour period, and that the two groups oscillated with opposite phases. This led them to conclude that the Synechococcus genome was regulated with circadian rhythmicity.

Function in Vitro
The circadian oscillators in eukaryotes that have been studied function using a negative feedback loop in which proteins inhibit their own transcription in a cycle that takes approximately 24 hours. This is known as a transcription-translation-derived oscillator (TTO) or a transcription-translation feedback loop (TTFL). Without a nucleus, prokaryotic cells must have a different mechanism of keeping circadian time. In 1998, Ishiura et al. determined that the KaiA, KaiB, and KaiC were responsible for the circadian negative feedback loop in Synechococcus by mapping 19 clock mutants to the genes for these three proteins.

An experiment by Nakajima et al., in 2005, was able to demonstrate the circadian oscillation of the Synechococcus KaiC phosphorylation in vitro. They did this by adding KaiA, KaiB, KaiC, and ATP into a test tube in the approximate ratio recorded in vivo. They then measured the levels of KaiC phosphorylation and found that it demonstrated circadian rhythmicity for three cycles without damping. This cycle was also temperature compensating. They also tested incubating mutant KaiC protein with KaiA, KaiB, and ATP. They found that the period of KaiC phosphorylation matched the intrinsic period of the cyanobacterium with the corresponding mutant genome. These results led them to conclude that KaiC phosphorylation is the basis for circadian rhythm generation in Synechococcus.

Cyanobacterial Clocks as Model Systems
Cyanobacteria are the simplest organisms that have been observed demonstrating circadian rhythms. The primitiveness and simplicity make the KaiC phosphorylation model invaluable to circadian rhythm research. While it is much simpler than models for eukaryotic circadian rhythm generators, the principles are largely the same. In both systems the circadian period is dependent on the interactions between proteins within the cell. With the cyanobacterial model, this can be demonstrated by the fact that when mutated versions of the clock proteins are used, the expressed period changes with the same shifts that are observed in the cyanobacteria with these mutations in their genomes.

This model of circadian rhythm generation also has implications for the study of circadian “evolutionary biology”. While the basic concept of a 24-hour feedback loop acting as a circadian clock is shared between these cyanobacteria and eukaryotic organisms, the actual proteins that are essential for these oscillations seem to be unrelated. This would indicate that a protein based circadian oscillation system evolved at least two separate times: once in cyanobacteria and then again in eukaryotes.

Circadian Oscillation
In cyanobacteria, techniques such as the insertion of luciferase genes and transcriptomic analysis have shown that the vast majority of the cyanobacterial genome is expressed in a rhythmic fashion. These genes fall under two broad categories: class 1 genes that are promoted by KaiBC and expressed at dusk as well as class 2 genes that are promoted by purF and are expressed at dawn. The 24-hour periodicity of these genes remains intact even as the cells divide very rapidly or very slowly. Therefore, the presence of circadian oscillators is critical for the expression of the cyanobacterial genome.

In cyanobacteria, the oscillator component of the circadian system appears to consists of three main clock proteins: KaiA, KaiB, and KaiC. These three components appear to act rhythmically in constant light and constant dark, despite the lack of photoelectric signals, such as the Sun, that organisms with circadian clocks typically rely on in order to entrain. Entrainment of this clock in cyanobacteria occurs as a result of the system’s sensitivity to ATP/ADP ratios within the cell and through the sensing of nighttime photosynthetic metabolites. Instead of acting like the transcription-translation feedback loop (TTFL) usually seen in eukaryotic clocks, the three proteins acted as a post-translational oscillator (PTO), which acted independently of the transcription and translation mechanisms. The rhythmicity of this system continued to be present when the system was both in vivo and in vitro.

In Synechococcus elongatus, whose clock proteins are often used as a model to study cyanobacterial circadian systems, the kaiABC gene cluster is responsible for the regulation of gene expression, chromosome compacting, and cell division timing. The system receives environmental signals through the use of proteins that are sensitive to the redox levels within the cell, such as CikA (circadian input kinase A) and LdpA (light-dependent protein A. In turn, the system’s output is primarily maintained by a two-component system that consists of two sensor histidine kinases, SasA (Synechococcus adaptive sensor A) and CikA, along with a response regulator, RpaA (regulator of phycobilisome association A). RpaA is a transcription factor that binds and regulates the expression of the promoter for the kaiABC cluster. This pathway acts as a TTFL that provides a secondary pathway for maintaining rhythmicity under certain conditions, such as conditions that include rapid growth or when kaiA overexpression eliminates the PTO due to kaiC hyperphosphorylation.

KaiABC Structure and Function
In the KaiABC complex, KaiA enhances the phosphorylation status of KaiC. In contrast, the presence of kaiB in the complex decreases the phosphorylation status of KaiC, suggesting that KaiB acts by antagonising the interaction between KaiA and KaiC. The activity of KaiA activates KaiBC expression, while KaiC represses it. The overall fold of the KaiA monomer is that of a four-helix bundle, which forms a dimer in the known structure. KaiA functions as a homodimer. Each monomer is composed of three functional domains: the N-terminal amplitude-amplifier domain, the central period-adjuster domain and the C-terminal clock-oscillator domain. The N-terminal domain of KaiA, from cyanobacteria, acts as a pseudo-receiver domain, and thus lacks the conserved aspartyl residue required for phosphotransfer in response regulators. The C-terminal domain is responsible for dimer formation, binding to KaiC, enhancing KaiC phosphorylation and thus playing a major role in generating circadian oscillations. There are some differences in proteins structures in the KaiA protein, however. For example, the KaiA protein from Anabaena sp. (strain PCC 7120) lacks the N-terminal CheY-like domain.

KaiB adopts an alpha-beta meander motif and can be found as a complex of functional monomers, usually forming a dimer or a tetramer.

KaiC belongs to a larger family of proteins; it performs autophosphorylation and acts as its own transcriptional repressor when it binds to ATP.

Also in the KaiC family is RadA/Sms, a highly conserved eubacterial protein that shares sequence similarity with both RecA strand transferase and lon protease. The RadA/Sms family are probable ATP-dependent proteases involved in both DNA repair and degradation of proteins, peptides, glycopeptides. They are classified in as non-peptidase homologues and unassigned peptidases in MEROPS peptidase family S16 (lon protease family, clan SJ). RadA/Sms is involved in recombination and recombinational repair, most likely involving the stabilisation or processing of branched DNA molecules or blocked replication forks because of its genetic redundancy with RecG and RuvABC.

Oscillation Pathways
The core of the cyanobacterial clock centers around one component of the kaiABC system: kaiC. KaiC acts as a autokinase, autophosphatase, and ATPase, and its rhythms of phosphorylation upon the effector proteins of this system are critical for establishing the circadian rhythm. It is a ring-shaped, homohexamer comprised of two linked P-loop ATPase domains (CI and CII) and also possesses a C-terminal protruding tail called the A-loop. KaiC displays phosphorylation rhythms at Ser-431 and Thr-432 residues that attenuate to a 24-hour period. These rhythms are regulated by kaiC’s interactions with kaiA and kaiB.

KaiA binds to the A-loops on CII, which results in kaiC’s autophosphorylation. The activity of kaiA draws a greater concentration of ATP to the area and stimulates the active site on kaiC. KaiB forms a complex with kaiC phosphorylated with Ser-431 which inactivates the bound kaiA. This binding, which occurs during the night and occurs at a slow rate, causes the inactivation of KaiA. When phosphorylation activity reaches a minimum, affinity between kaiB and kaiC decreases. KaiA is then free to bind to the CII domain of kaiC once again.

The input/output pathway that contains sasA, cikA, and rpaA is responsible for regulating patterns in cell division and gene expression. SasA possesses a N-terminal domain that resembles kaiB, which promotes sasA’s phosphorylation. SasA then performs a phototransfer to the response regulator, rpaA. This mechanism by sasA activates rpaA. Upon phosphorylation, rpaA binds to DNA at numerous locations within the cyanobacterial genome. CikA, a histidine kinase, is responsible for removing the phosphoryl groups that are added by sasA. The activity of both sasA and cikA are controlled by the circadian oscillator. SasA and cikA antagonize each other to generate rhythmic rpaA phosphorylation, resulting in a mechanism that is able to receive circadian inputs and create circadian outputs, key traits in establishing circadian genetic expression in cyanobacteria.