dCLOCK Is Present in Limiting Amounts and Likely Mediates Daily Interactions between the dCLOCK–CYC Transcription Factor and the PER–TIM Complex

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Abstract
InDrosophila melanogasterfour circadian clock proteins termed PERIOD (PER), TIMELESS (TIM), dCLOCK (dCLK), and CYCLE (CYC/dBMAL1) function in a transcriptional feedback loop that is a core element of the oscillator mechanism. dCLK and CYC are members of the basic helix-loop-helix (bHLH)/PAS (PER-ARNT-SIM) superfamily of transcription factors and are required for high-level expression ofperandtimand repression ofdClk, whereas PER and TIM inhibit dCLK–CYC-mediated transcription and lead to the activation ofdClk. To understand further the dynamic regulation within the circadian oscillator mechanism, we biochemically characterizedin vivo-produced CYC, determined the interactions of the four clock proteins, and calculated their absolute levels as a function of time. Our results indicate that throughout a daily cycle the majority of the dCLK present in adult heads stably interacts with CYC, indicating that CYC is the primaryin vivopartner of dCLK. dCLK–CYC dimers are bound by PER and TIM during the late evening and early morning, suggesting the formation of a tetrameric complex with impaired transcriptional activity. Although dCLK is present in limiting amounts and CYC is by far the most abundant of the four clock proteins that have been examined, PER and TIM appear to interact preferentially with dCLK. Our results suggest that dCLK is the main component regulating the daily abundance of transcriptionally active dCLK–CYC complexes.