Molecular Clocks

"Every indication of contrivance, every manifestation of design, which existed in the watch, exists in the works of nature; with the difference, on the side of nature, of being greater or more, and that in a degree which exceeds all computation" -William Paley (TBW:4-5)

Last Updated:
October 6, 2005

1. Researchers Unwind Secrets of Biological Clocks

"COLLEGE STATION, January 30, 2003 – It may be only pond scum, the sort of green gunk that clogs lakes and floats in on the tides. But inside, a clock is quietly ticking.

Even this lowly one-celled bacterium has a biological clock, the sophisticated internal timing device that governs the daily rhythms of people, animals and plants, says Susan Golden, a biology professor at Texas A&M University. The university’s Department of Biology is a leader in unraveling the mysteries of biological clocks, research that eventually could lead to cures for sleep and mood disorders, as well as other medical problems.

Golden and her colleagues also study the biological clocks of birds, rats and fungi, but it was the bacterium known as Synechococcus elongatus that yielded the latest revelation: the first structural model of part of the clockworks.

The researchers used magnetic-resonance imaging to limn the structure of one of the proteins produced by a trio of genes that function like the turning gears in a wristwatch. The protein in this microscopic “timing-input device” takes in and transmits environmental signals to adjust the clock for seasonal changes in day and night, according to the model, published recently in Proceedings of the National Academy of Science.

Although scientists do not know whether the same genetic scheme also pertains to biological clocks in people, Golden said, revealing the structural diagram is “a very important step” to learning how the clocks actually work.

It was no small trick to produce a model from a one-celled organism so simple that no one can even see just how it acts out its daily rhythms of life.

The organism doesn’t even have a nucleus, after all, much less a discernable daily routine. So Golden, a molecular biologist who has devised ways to genetically manipulate Synechococcus, rigged a sample with a tiny “light meter” that reflected daily changes inside the organism. She used luciferase, the same enzyme that illuminates fireflies, to make a “reporter gene” that literally shed light on its rhythms. “It tells us what the clock is doing,” Golden said.

The magnetic-resonance imaging work that helped to reveal the three-dimensional structure was performed in the laboratory of Andy LiWang, an assistant professor of biochemistry and biophysics at Texas A&M. Related research on the two other genes in the trio that make up the timing device (i.e., just the part we have structure for has an input role, but all three are main gears of the clock) was conducted at Vanderbilt University and Nagoya University in Japan.

Researchers at Texas A&M, including students, are continuing to test genes in Synechococcus to see what others might also make up the biological clock, Golden said.

This story has been adapted from a news release issued by Texas A&M University ."

2. Meshing the Gears of the Cyanobacterial Clock

"Susan S. Golden *

Center for Research on Biological Clocks, Department of Biology, Texas A&M University, College Station, TX 77843-3258

Aphysiological black box is to a biologist what an ornately decorated package is to a small child: a mysterious treasure that promises delightful toys within. With fitting élan, a small community of scientists has ripped open the packaging of the cyanobacterial circadian clock, compiled the parts list, examined the gears, and begun to piece together the mechanism. Over the past 2 years, the 3D molecular structures have been solved for the core components of the cyanobacterial circadian clock: KaiA, KaiB, and KaiC (16). In a surprisingly literal analogy to mechanical timepieces, the protein that seems to be at the heart of the clock mechanism, KaiC, forms a hexameric ring that even looks like a cog: the escape wheel, perhaps (5, 7, 8). Previous work has shown that KaiC has an autophosphorylation activity, and that the presence of KaiA and KaiB modulates the extent to which KaiC is phosphorylated (6, 9). In this issue of PNAS, Nishiwaki et al. (10) biochemically identify two amino acid residues on KaiC to which phosphoryl groups covalently attach, and show the necessity in vivo of a phosphorylation-competent residue at these positions. By searching the crystal structure for evidence of phosphorylated sites, Xu et al. (11) pinpoint a third residue that may "borrow" the phosphoryl group dynamically. Together, their work contributes richly to our understanding of what makes the gears mesh and turn to crank out a 24-h timing circuit.

The emerging model of the cyanobacterial circadian oscillator differs markedly from those that have been proposed for various eukaryotic systems (12, 13). In the latter schemes, many of the central components are either transcription factors that directly stimulate the genes encoding other clock components, or antagonists of the transcription factors that block their ability to carry out such stimulation. This basic yin–yang of mutual negative regulation is embellished by kinases that affect the stability of factors, heterodimer formation, movement of dimers from the cytoplasm to the nucleus, and exchange of partners inside the nucleus. Central to the models is the resulting undulation of transcription from clock component genes. However, the underlying timing mechanism in cyanobacteria does not depend on transcription of the kai genes by specific factors, because heterologous promoters can substitute admirably for expression of KaiC (14, 15). In addition, expression of the three known clock components oscillates in a shared phase, precluding their function as a teeter–totter-style interlocked transcription feedback loop, although overexpression of KaiA or KaiC stimulates or represses, respectively, kaiBC expression. The cyanobacterial clock, as described for the model organism Synechococcus elongatus, does have some features reminiscent of the posttranslational aspects of eukaryotic clocks: key components oscillate in abundance with one cycle per day, and the phosphorylation state of a crucial factor changes rhythmically as well.

The cyanobacterial clock assembles and disassembles during the course of a day, defining the circadian period.

What, then, is the fundamental biochemical event that takes a day to complete in cyanobacteria? Unlike the tag-team relay of eukaryotic clock parts, the cyanobacterial clock components engage in a group hug (13). Analysis of the sizes of complexes in which KaiA, KaiB, KaiC, and a closely associated kinase, SasA, are found throughout the day shows that late at night they are all in a very high molecular-weight complex, presumably a shared complex (16). Because each of these components (at minimum) is a dimer, KaiC is known to be a hexamer, and other proteins may be present as well, the cyanobacterial clock can be thought of as an organelle unto itself: a "periodosome" that assembles and disassembles during the course of a day, defining the circadian period.

It is the assembly of the periodosome that is likely to be influenced by the phosphorylation state of KaiC. Nishiwaki et al. (10) identified serine 431 and threonine 432 as the residues that become phosphorylated when KaiC is incubated in vitro with ATP. Xu et al. (11) recognized the same residues based on density differences between phosphorylated and nonphosphorylated KaiC subunits and reasoned that threonine 426, which faces serine 431, is near enough to hydrogen bond with the phosphoryl group and perhaps serve as an alternate ligand. Mutation of any of these three residues to alanine, which is not a substrate for phosphorylation, abolished the circadian rhythms of gene expression in S. elongatus. However, the hexamerization of KaiC was not affected, even in a double or triple mutant that shows no phosphorylation of KaiC in vivo.

An important difference in the S431A:T432A double mutant relative to wild type is the complete inability to coimmunoprecipitate, with KaiC, the other proteins that are usually part of the periodosome (10) (Fig. 1). KaiA and KaiB are known to directly interact with KaiC, to be part of the high molecular-weight complex that forms during the night, and to affect the phosphorylation rate of KaiC (positively and negatively, respectively) (13). The new data indicate that specific phosphorylation of KaiC is necessary to allow the other Kai proteins, in turn, to exert their influence. The extent to which KaiC is phosphorylated varies during the circadian cycle, as may the ratios of particular phosphorylated states. It is easy to imagine that each phosphoryl modification tweaks the conformation, shifting the KaiC landscape to enhance or exclude access to a heterologous partner; the heterotypic interaction, reciprocally, changes the receptivity of KaiC ligands to phosphorylation. Some phosphorylation-dependent changes in the hexamer itself can be inferred from the crystal structure (11). A glimpse into the conformational changes that Kai heterotypic interactions impart is evident in the rotation of the C-terminal dimeric domain of KaiA when it binds a specific peptide of KaiC (4).

Specific phosphorylation of KaiC is necessary to allow the other Kai proteins to exert their influence.

Clearly, the regulated phosphorylation of KaiC and the ability to form the periodosome comprise steps in the time delay that accumulates 24-h time in the cell. Less clear is what the complex does when it forms. Both groups show that overexpression of nonphosphorylating mutant KaiC variants causes suppression of expression from the kaiBC promoter, although the reports differ as to whether this is a transient effect. The mechanism of negative autoregulation of kaiC is not known, but it appears to be a global effect on gene expression rather than a specific feedback (14). Direct interaction of KaiC with the cyanobacterial chromosome has been proposed but not demonstrated.

Mutation to alter the residues that KaiC will autophosphorylate in vitro resulted in nonphosphorylated KaiC in vivo (10, 11). This result might suggest that there are no external kinases that act on KaiC. However, it is equally possible that autophosphorylation is a prerequisite to allow access to a KaiC kinase, just as it is needed for interaction with KaiA, KaiB, and SasA. Identification of other potential components of the periodosome, intracellular localization of the clock parts, and elucidation of other potential modifications all may yield gears that are required to smoothly tick away the time and ensure that daughter cells do not run fast or slow.

The cyanobacterial clock box, no longer black, is a chest filled with bioluminescence and attractive toys. Putting together the pieces to design a clock is a tedious task, but S. elongatus is a gracious host, and the guests at the party are hard at work.


See companion articles on pages 13927 and 13933.

* E-mail: .

© 2004 by The National Academy of Sciences of the USA


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Researchers Unwind Secrets of Biological Clocks

Meshing the Gears of the Cyanobacterial Clock





































































































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