dCLOCK is present in limiting amounts and likely mediates daily interactions between the dCLOCK-CYC transcription factor and the PER-TIM complex

In Drosophila melanogaster four circadian clock proteins termed PERIOD (PER ), TIMELESS (TIM), dCLOCK (dCLK), and CYCLE (CYC/dBMAL1) function in a tran scriptional feedback loop that is a core element of the oscillator mechanis m. dCLK and CYC are members of the basic helix-loop-helix (bHLH)/PAS (PER-A RNT-SIM) superfamily of transcription factors and are required for high-lev el expression of per and tim and repression of dClk, whereas PER and TIM in hibit dCLK-CYC-mediated transcription and lead to the activation of dClk. T o understand further the dynamic regulation within the circadian oscillator mechanism, we biochemically characterized in 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 wit h CYC, indicating that CYC is the primary in vivo partner 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 transcripti onal activity. Although dCLK is present in limiting amounts and CYC is by f ar the most abundant of the four clock proteins that have been examined, PE R and TIM appear to interact preferentially with dCLK. Our results suggest that dCLK is the main component regulating the daily abundance of transcrip tionally active dCLK-CYC complexes.