Circadian Rhythm Set by Pairing of Two Proteins
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The molecular control of the daily cycle known as circadian rhythm lies in the pairing of two proteins, National Science Foundation (NSF) scientists report in a trio of papers in the November 3 issue of the journal Science. The findings, derived from fruit fly studies, promise to help scientists better understand human, animal and plant circadian rhythms. These rhythms influence cell and body biochemistry, health, aging and behavior.
"Our data show the setting and running of the daily body clock comes from the delicate affinity of two proteins," says Michael Young of the National Science Foundation (NSF) Science and Technology Center for Biological Timing in Charlottesville, Virginia, who is also affiliated with Rockefeller University in New York.
Like people, fruit flies have daily rhythms lasting approximately 24 hours. In 1971, scientists at the California Institute of Technology discovered that a fly gene dubbed period (per) was involved in the clock, but exactly how it worked was unknown. In 1984, Young and his collaborators at Rockefeller University and a second group of scientists at Brandeis University cloned the per gene and characterized the protein it makes. In 1994, Young's group identified a second clock gene, dubbed timeless (tim).
In the new studies, Young and collaborators from Rockefeller, the University of Pennsylvania and Harvard Medical School cloned the tim gene, determined the order of nucleic acids in its DNA structure and characterized the protein it makes, TIM. By investigating what happens when tim and per are damaged in mutated flies, the researchers also established how the TIM and PER proteins together set the body clock.
"The tim and per genes, through the proteins they make, have a true partnership in operating the body's clock," says Young. "We found that part of the TIM protein binds to the PER protein. Once joined, the proteins enter the cell nucleus, a process that sets the time and duration of the circadian cycle."
All cells in the fly have per and tim genes, but the cells in the fly's brain set the body's clock. The two genes become active at midday. In the cell's nucleus, the genes' DNA code is transcribed into two RNA molecules, per RNA and tim RNA, which accumulate over several hours in the cell.
At dusk, the levels of RNAs peak and only then does the cell use the RNAs to stockpile PER and TIM proteins. In the evening, the proteins join and cross into the cell's nucleus. About four hours before dawn, the PER and, presumably, TIM proteins in the nucleus reach their maximum amounts, an achievement that signals the per and tim genes to stop making the RNA. Near dawn, the nuclear proteins begin disintegrating, and the cycle begins again. Throughout the daylight hours the per and tim genes produce new RNA to make replacement proteins.
The pace of the clock appears to stem from the gradual, coordinated accumulation of the tim and per RNAs during several hours, as well as from the attraction of the PER and TIM proteins for each other, Young reports.
"The PER and TIM proteins have an affinity for each other, but it is not a strong link. Only if the two proteins are available in sufficient quantities do they begin to bind. Most importantly, the proteins can only survive and enter the cell nucleus when they are bound to each other. Therefore, about six to eight hours lapse between the time of peak RNA accumulation, which occurs around dusk, and the peak in the nuclear protein levels, shortly before dawn."
Additional mechanisms and other as-yet-unidentified proteins also may influence the interaction between the PER and TIM proteins, which could affect the timing, Young adds. For example, scientists know that light affects circadian rhythms. Young also notes that evidence exists that the PER/TIM protein union is affected by light. This sensitivity may help explain how body clocks are reset after a period of jet lag that occurs as a traveler crosses time zones.
In addition, the scientists are searching for the genes of the human body clock. "In general, the genes that control fundamental body mechanisms are passed on in evolution," Young explains. "Now that we know the mechanisms in the fly's body clock that produce the TIM and PER proteins, and the feedback loops involved, we expect to find a similar process in the body clocks of humans."
In humans, daily circadian rhythms underlie many functions, including the sleep/wake cycle, body temperature, mental alertness, pain sensitivity and hormone production. In natural conditions, many rhythms have a 24-hour period related to sunlight, but though light can affect the rhythm, it does not cause the cycle. In fact, in the absence of light or other environmental clues, rhythms continue and most adapt to periods slightly longer or shorter than 24 hours, Young notes.
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