Several genes, expressed in neurons of the suprachiasmatic nucleus (SCN), are activated or inhibited in a cyclical pattern over the span of a day. These oscillations are the molecular "gears" of the biological clock regulating our 24-hour rhythms. A key feature of these molecular oscillations is the negative feedback loop formed when the protein product of a gene actually turns off production of more protein. The following animation shows the molecular interactions involved in the negative feedback loop responsible for circadian rhythms in mammals.
Part 1: The role of Per and Cry in the mammalian molecular model
Mammalian SCN neurons contain three types of Period genes (Per1, Per2, and Per3) and two types of cryptochrome genes (Cry1 and Cry2). Although the role of each gene is slightly different, this animation features one Per and one Cry gene for clarity.
Per and Cry genes in the nucleus are activated by the binding of the proteins BMAL1 and CLOCK (positive activators) to their promoters. (BMAL1 and CLOCK are the mammalian equivalents of Drosophila CYCLE and CLOCK.) Transcriptional activation results in the production of mRNA, which exits the nucleus through nuclear pores and is translated into protein by the ribosomes.
PER protein is susceptible to degradation (pink) unless it forms a dimer (red). A dimer is a pair of molecules. PER/PER and PER/CRY dimers translocate into the nucleus. The dimers interact with BMAL1/CLOCK to block activation. A negative feedback loop is created: PER and CRY proteins (negative regulators) block transcription by their own genes.
Over time, the dimers degrade and are replaced by other dimers. Eventually, too few dimers are available to block activation because no more proteins are being made. Inhibition is relieved, and transcriptional activation begins again.
Part 2: Three roles of casein kinase 1 epsilon (CK1e)
Mammalian CK1e is a critical regulator of circadian oscillations. This kinase enzyme is the mammalian equivalent of the Drosophila doubletime molecule.
CK1e in the cytoplasm phosphorylates susceptible PER proteins (pink), making them less stable and eventually resulting in their degradation. Second, CK1e is involved in the translocation of dimers from the cytoplasm to the nucleus. Third, CK1e plays a role in the degradation of the inhibitory complex in the nucleus formed by PER and CRY dimers. Following degradation of these dimers, transcription can begin again.
Part 3: Cytoplasmic activity of mutant CK1e results in a shortened period
This animation illustrates how a mutation in CK1e results in a change in the timing of production of proteins that influence circadian behaviors. Specifically, hamsters homozygous for the tau mutation—that is, they have two copies of the mutated gene—have a shortened circadian period. Instead of following a 24-hour clock, these hamsters operate on a 20-hour clock. A small change to the CK1e protein causes CK1e to become much less effective in phosphorylating PER proteins, so fewer PER molecules degrade.
As a result, PER dimers accumulate faster in the cytoplasm of a mutant hamster's SCN neurons. With faster accumulation, translocation of the inhibitory complex into the nucleus occurs sooner. Therefore, inhibition of per gene transcription occurs earlier in the mutant hamster than in the wild type. The final effect is that the mutant hamster has a shortened period relative to that of the wild-type hamster.
Part 4: Mutant human Period2 gene results in shortened sleep cycle
Within neurons in the brain's suprachiasmatic nucleus, a complex orchestration of molecular changes regulates our biological clock. Similar to the clock in other mammals, the human clock is thought to be based around a negative feedback loop in which the protein product of a gene turns off further gene transcription and protein synthesis. This cycle of genes turning on and off is correlated with the normal cycle of sleeping and waking.
The functions of many circadian genes and proteins have been demonstrated in Drosophila, hamsters, and mice. As the functions and structures of these nonhuman genes are clarified, researchers have searched for similar genes in the human genome. Indeed, many structurally similar genes are present in humans. However, to confirm the functional relevance of human genes in circadian rhythmicity, researchers examined circadian genes in individuals who have an abnormally short sleep-wake cycle.
Genetic analysis revealed that the human Period2 gene (hPer2) in individuals with familial advanced sleep phase syndrome (FASPS) contains a single base-pair mutation. This mutation alters the site in the hPER2 protein through which casein kinase 1 epsilon (CK1e) acts. Normally, CK1e would add a phosphate group to the hPER2 protein. Phosphate groups target the protein for degradation. However, because of the altered site, the mutant hPER2 protein is less susceptible to degradation.
This animation presents a possible model of the molecular interactions in the human biological clock. The negative feedback loop in normal individuals ("Wild type") is compared with alterations that likely occur in individuals with FASPS ("Mutant hPER2"). Specifically, the hPer2 gene produces hPER2 protein that is less susceptible to CK1e degradation than that of the wild-type. With less degradation of hPER2 in the mutant, hPER2 accumulates more rapidly in the cytoplasm. After entering the nucleus, these hPER2 proteins turn off hPer2 gene transcription. The end result is a sleep cycle that is shorter in the mutant than in the wild type.
Mammalian Molecular Model Background
Living organisms have evolved internal timekeeping mechanisms to synchronize behavior and physiology with the cycles of day and night. These biological clocks have been found in organisms as diverse as fungi, fruit flies, hamsters, and humans.
This animation demonstrates the molecular control of circadian rhythms within the neurons of the suprachiasmatic nucleus.
Species-specific forms of the period gene and its protein product PER are essential components of the negative feedback loop that regulates circadian rhythms. The Drosophila regulatory enzyme doubletime is the equivalent of the mammalian casein kinase 1 epsilon enzyme. The same transcription factors that turn on gene transcription in Drosophila (CYCLE and CLOCK) also turn on gene transcription in mammals (BMAL1 and CLOCK). (BMAL1 is identical to CYCLE; the same molecule in different organisms often has a different name.) While cryptochrome is an essential molecular regulator for both Drosophila and mammalian circadian rhythms, its function is quite different in these two organisms. Cryptochrome in Drosophila is directly responsive to light input; light can pass through the exoskeleton of Drosophila and enter neurons, where it produces a conformational change in cryptochrome. This activated cryptochrome then effects the degradation of TIM proteins in the nucleus. In contrast, the cryptochrome gene in mammals acts in concert with the period gene in circadian rhythm regulation through the negative feedback loop. An active area of research is the examination of how light affects mammalian circadian clock genes.
This animation was designed in conjunction with HHMI's 2000 Holiday Lectures on Science, Clockwork Genes: Discoveries in Biological Time (www.holidaylectures.org).
Mammalian Molecular Model Tips
The animations in this section have a wide variety of classroom applications. Use the tips below to get started but look for more specific teaching tips in the near future. Please tell us how you are using the animations in your classroom by sending an e-mail to firstname.lastname@example.org.
Use the animations to make abstract scientific ideas visible and concrete.
Explain important scientific principles through the animations. For example, the biological clocks animations can be used to demonstrate the fundamentals of transcription and translation.
Make sure that students learn the material by repeating sections of the animations as often as you think necessary to reinforce underlying scientific principles. You can start, restart, and play back sections of the animations.
Urge students to use the animations in accordance with their own learning styles. Students who are more visually oriented can watch the animations first and read the text later, while others might prefer to read the explanations first and then view the graphics.
Incorporate the animations into Web-based learning modules that you create to supplement your classroom curricula.
Encourage students to incorporate the animations into their own Web-based projects.
Mammalian Molecular Model References
1. Bear, M.F., Connors, B.W., and Paradiso, M.A. Neuroscience: exploring the brain. Baltimore: Williams and Wilkins, 1996.
2. Herzog, E.D., Takahashi, J.S., and Block, G.D. 1998. Clock controls circadian period in isolated suprachiasmatic nucleus neurons. Nature Neuroscience 1:708-713.
3. Lydic, R., Albers, H.E., Tepper, B., and Moore-Ede, M.C. 1982. Three-dimensional structure of the mammalian suprachiasmatic nuclei: a comparative study of five species. J. Comp. Neurol. 204:225-237.
4. van den Pol, A. 1980. Hypothalamic suprachiasmatic nucleus: intrinsic anatomy. J. Comp. Neurol. 191:661-702.