The Drosophila molecular circadian clock consists of two main interlocked sets of transcription/translation feedback loops. These both contain protein and mRNA components that cycle in abundance and subcellular localisation with a near 24 hour period. Within the core components of the clock pathway, both loops drive rhythmic transcription in the opposite phase to each other.
The pathways shown below are most representative of those occurring in key clock neurons within the brains of adult flies kept under standard 12 hour light:12 hour dark cycles at 25°C. Differences in one or more of the biochemical steps shown here and/or reactions not shown here can occur in different brain clock neurons (about 150 total cells per adult brain) and within the same clock cells as a function of changes in environmental conditions, such as day-length or temperature.
Starting from midday and continuing through to early night, the per/tim feedback loop is initiated when the basic-helix-loop-helix/PAS (PER-ARNT-SIM) domain transcription factors, Clock (CLK) and Cycle (CYC), form heterodimers and bind E-box regulatory elements to activate transcription of target genes per, tim, vri, and Pdp1. The levels of per and tim mRNA transcripts peak early in the night, whereas Period (PER) and Timeless (TIM) proteins do not reach peak abundance levels until mid-to-late evening. TIM stays at low levels at this point because it is destabilised by light. PER is phosphorylated by Discs overgrown (DCO) kinase aka Double-time (DBT) and Casein kinase II (CK2). Without TIM, which stabilises it, PER is targeted for degradation by the 26S proteasome via an interaction with the F-box containing protein, supernumerary limbs (SLMB). In addition, PER is stabilised by the protein phosphatases PP2A and PP1 while TIM is similarly stabilised by PP1 phosphatase. The coordinated action of both kinases and phosphatases, during this part of the day, keep PER at low levels and in a hypophosphorylated state, as hyperphosphorylated PER is removed due to low TIM levels.
After dusk during the early night, per and tim mRNA transcripts reach peak levels. TIM abundance begins to increase in the dark and forms a complex with PER. This, along with PP1 phosphatase, stabilises PER despite its continued phosphorylation by kinases. As a result PER and TIM accumulate to high levels during the middle of the night. TIM is then phosphorylated by CK2. PER and TIM dissociate, PER (in complex with DCO) enters the nucleus followed by TIM, a few hours later. Once in the nucleus PER and TIM reform their heterodimer and bind to the CLK:CYC heterodimer, so repressing the transcription of per, tim, vri, and Pdp1. The CLK:CYC heterodimer is removed from the E-boxes and sequestered in a stable complex with PER and TIM to reinforce the transcription inhibition. In addition, DCO bound to PER phosphorylates CLK. Towards the end of the night PER and TIM proteins are at their peak level.
At dawn, the introduction of light recalibrates the circadian clock. The blue-light photoreceptor cryptochrome (CRY) experiences a light-induced conformational change which forms a complex with hyperphosphorylated TIM, which leads to both TIM and CRY degradation by the 26S proteasome via an interaction with the F-box containing protein Jetlag (JET). PER and CLK, hyperphosphorylated by DCO, the former no longer protected by TIM, are also degraded during the early morning. PER levels fall to their lowest level around midday, however, CLK levels remain constant since hypophosphorylated CLK is generated from new CLK protein expression or dephosphorylation of hyperphosphorylated CLK by PP2A protein phosphatase. Hypophosphorylated CLK then forms a heterodimer with CYC, binds to E-boxes of its target genes and a new cycle of per, tim, vri, and Pdp1 transcription is initiated. There is an alternate view that CLK binds to CYC then CLK is phosphorylated in the cytosol. This promotes nuclear entry followed by further CLK phosphorylation. However, this string of events at present remain controversial and are not included in the current model.
There is a second feedback loop, the Clk loop, interlocked with the per/tim feedback loop. As mentioned above, at midday CLK:CYC heterodimers bind to the E-boxes and activate transcription of the target genes vri and Pdp1, which lead to the production of the proteins Vrille (VRI) and PAR domain protein-1 (PDP1) respectively. PDP1 activates transcription of Clk and cry genes, while VRI represses it, by competitively binding to regulatory sequences called VRI/PDP1 (V/P) boxes. Although vri mRNA accumulates in phase with per and tim mRNAs, Pdp1 accumulation is delayed by several hours. In contrast to the delayed accumulation of PER and TIM protein levels, VRI protein levels rise in sync with vri mRNA. Between midday and dusk, VRI levels rise, and the superior VRI/PDP1 ratio means it predominantly binds to the V/P boxes to repress Clk and cry transcription. VRI levels reach their peak during the early night after dusk coinciding with minimal levels of Clk and cry mRNAs. However, CRY protein levels build slowly as it is relatively stable in the dark.
PDP1 levels reach their peak between mid to late night. VRI levels decline during this time due to TIM:PER influenced repression of vri transcription. The decreasing ratio of VRI/PDP1 levels favours binding of PDP1 to the V/P boxes resulting in activation of Clk and cry transcription. PDP1 protein levels start to decline during the late night and are low by the early morning. However, despite this, PDP1 continues to activate Clk and cry transcription until midday when VRI protein levels are sufficient, after the next cycle of CLK:CYC mediated transcription, to take over the binding of the V/P boxes and repress transcription.
Recently, CLK:CYC transcription of some new target genes and their associated feedback loops have come to light such as the cwo gene and its protein product clockwork orange (CWO). However, there is confusion as to whether CWO, which binds to the E-boxes of per, tim, vri, and Pdp1 activates or represses their transcription. There are likely to be many more of these secondary transcription loops connected to the core clock mechanism to add yet more modes of regulation.