Like many other organisms, the fruit fly Drosophila melanogaster operates on a 24-hour schedule maintained by environmental input to an internal body clock. The molecular basis of the clock relies on oscillations in the activation of particular genes at certain times of the day. The key feature of these molecular oscillations is a negative feedback loop in which the protein products of genes actually turn off production of more protein. This process is possible in all cells of Drosophila; however, the highest concentrations of the essential molecules are found in lateral neurons of the central nervous system. These lateral neurons, or pacemaker cells, are the Drosophila equivalent of mammalian neurons in the suprachiasmatic nucleus. Watch these animations display the dynamic orchestration of the molecular events of the Drosophila biological clock.
Part 1: Essentials of the negative feedback loop
The negative feedback loop that forms the basis of the Drosophila molecular clock occurs at the level of gene transcription.
The activated period (per) gene in the nucleus of the cell is seen transcribing messenger RNA (mRNA) molecules. As the animation begins, per mRNA moves to the cytoplasm, where ribosomes translate the mRNA into PERIOD (PER) protein molecules. Some PER molecules (shown in pink) degrade shortly after synthesis; others (shown in red) are stable and accumulate in the cytoplasm.
PER protein levels reach a maximum during the middle of the night. At that point, the stable PER molecules enter the nucleus. Inside the nucleus, the PER protein inhibits transcription of its own gene. The per gene turns black to indicate that transcription is repressed.
As the sun rises, PER molecules become susceptible to degradation (shown in pink). Over the course of several hours, all PER protein disappears. In the absence of PER, transcription of the per gene begins again.
Like most genes, the DNA sequence of the per gene contains an upstream regulatory region called the promoter (left red rectangle), followed by the DNA template for mRNA transcription (right red rectangle). For the per gene to be transcribed, two proteins, CYCLE (CYC) and CLOCK, must bind to a DNA region called the E-box in the per gene promoter.
As the animation begins, it is night. The CYC/CLOCK complex is bound to the promoter, and the per gene is transcribed. Transcription is repressed when PER protein molecules interact directly with the CYC/CLOCK complex. After the sun rises, however, PER molecules degrade, thereby releasing the repression of the CYC/CLOCK complex. As a result, per gene transcription resumes.
Like the per gene, transcription of the timeless (tim) gene is activated by the proteins CYC and CLOCK. Following transcription, tim and per messenger RNA (mRNA) molecules are translated in the cytoplasm to make TIM and PER proteins. TIM and PER proteins bind to one another to form a heterodimer (a molecule formed by joining two nonidentical molecules). The formation of a complex with TIM protects PER from rapid degradation.
PER/TIM complexes enter the nucleus, where they directly interact with CYC/CLOCK complexes. This interaction represses transcription of the tim and per genes. As the sun rises, light causes rapid degradation of TIM. Without TIM as a stabilizing partner, PER also degrades. The repression of CYC/CLOCK is thereby released, and transcription resumes.
The Drosophila doubletime protein, which is found both in the cytoplasm and nucleus, is homologous (evolutionarily closely related) to mammalian casein kinase 1 epsilon. Kinases are enzymes that add phosphate groups to molecules.
The addition of phosphate groups to the PER protein accelerates its degradation. As PER protein is synthesized in the cytoplasm, doubletime (shown as a triangle) causes the degradation of PER proteins (pink). However, PER proteins that have formed complexes with TIM proteins are resistant to degradation by doubletime. PER/TIM complexes enter the nucleus and some interact with CYC/CLOCK, resulting in the repression of transcription of the per and tim genes.
As the sun rises, light causes a conformational (shape) change in the cryptochrome protein, thereby activating it. (Cryptochrome is shown as an orange diamond when it is inactive and changes into a circle when it becomes active.) Activated cryptochrome interacts with TIM, causing it to degrade. Without the stabilization provided by TIM, PER proteins become susceptible to degradation by the doubletime protein in the nucleus.
The degradation of PER/TIM results in the release of repression, and transcription of the per and tim genes begins once again.
A specific mutation in the doubletime kinase molecule results in a fruit fly with a period of about 28 hours. Watch this animation display how a less-effective doubletime molecule results in a lengthened period.
Doubletime phosphorylates PER monomers (single molecules) in the cytoplasm, resulting in PER degradation. This process in the mutant is less effective than that in the wild type (shown by the mutant doubletime "firing twice" to result in PER degradation, while wild type doubletime "fires" only once). Nonetheless, the accumulation of PER/TIM heterodimers in the cytoplasm is comparable to that observed in wild-type flies. (A heterodimer is a molecule formed by joining two nonidentical molecules.)
PER/TIM heterodimers block transcription. Light induces a conformational change in cryptochrome. Cryptochrome degrades TIM.
At this point, the timing of PER degradation differs in the mutant (left screen) and wild type (right screen). Mutant doubletime degrades PER but at a slower rate than that of wild type. As a result, the per gene's release from repression occurs later in the mutant; in turn, per and tim genes are activated later. The resulting effect on the fruit fly's circadian rhythm is a lengthened period.
Drosophila 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.
To understand how normal and mutant genes influence circadian rhythms at the molecular level, this animation demonstrates molecular interactions within a single cell in the nervous system of the fruit fly. The animation is divided into parts that progressively increase in complexity, beginning with basic principles of transcription, translation, and a negative feedback loop. The molecular changes are correlated with day-night cycles. Finally, the effects of mutant molecules on the length, or period, of the daily cycle are shown.
Consistent symbols are used in both Drosophila and the mammalian molecular model animations. 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 similar to the mammalian casein kinase 1 epsilon enzyme. The transcription factors that turn on gene transcription in Drosophila (CYCLE and CLOCK) are close relatives of gene transcription factors in mammals (BMAL1 and CLOCK). (BMAL1 is homologous 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.
Drosophila Molecular Model Teaching 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.
Drosophila Molecular Model Credits
Director: Dennis Liu, Ph.D.
Scientific Direction: Michael Rosbash, Ph.D.
Scientific Content: Donna Messersmith, Ph.D.
Animators: Chris Vargas, Eric Keller