TIMELESS-dependent positive and negative autoregulation in the Drosophila circadian clock.

The timeless protein (TIM) is a central component of the circadian pacemaker machinery of the fruitfly Drosophila melanogaster. Both TIM and its partner protein, the period protein PER, show robust circadian oscillations in mRNA and protein levels. Yet the role of TIM in the rhythm generation mechanism is largely unknown. To analyze TIM function, we constructed transgenic flies that carry a heat shock-inducible copy of the timeless gene (tim) in an arrhythmic tim loss-of-function genetic background. When heat shocked, TIM levels in these flies rapidly increased and initiated a molecular cycle of PER accumulation and processing with dynamics very similar to the PER cycle observed in wild-type flies. Analysis of period (per) mRNA levels and transcription uncovered a novel role for TIM in clock regulation: TIM increases per mRNA levels through a post-transcriptional mechanism. Our results suggest positive as well as negative autoregulation in the Drosophila circadian clock.