In the same study, we investigated translational control as a possible mechanism for independent control of PER cycling. We found no evidence for translational control of per RNA, suggesting that the cycling of PER is due solely to control of stability. We have also characterized the mechanisms that drive cycling of tim RNA, using a combination of biochemical and transgenic approaches. Since it is likely that the cycling of TIM is also independent of its RNA, we are addressing the importance of protein cycling for both PER and TIM through other experiments. Reasoning that protein cycling could only be eliminated by saturating endogenous turnover mechanisms, we have constitutively overexpressed both PER and TIM in transgenic flies.
Experiments are under way to determine the role of protein cycling in behavioral rhythms.
Our hypothesis is that behavioral rhythms require rhythmic effects of the PER and TIM proteins on the transcription of downstream genes in the circadian system. Thus we postulate that protein cycling or, more specifically, rhythmic nuclear expression of the two proteins is critical for rhythmic behavior. These studies have provided insights into the mechanisms underlying different aspects of the feedback loop and have also addressed models for resetting of the circadian clock by light. We and others demonstrated previously that levels of TIM are reduced in response to light. Our subsequent dose-response experiments showed that the TIM response to light correlates with the behavioral response.