Abstract

--- - Circadian clocks temporally coordinate daily organismal biology over the 24-h cycle. Their molecular design, preserved between fungi and animals, is based on a core-oscillator composed of a one-step transcriptional-translational-negative-feedback-loop (TTFL). To test whether this evolutionarily conserved TTFL architecture is the only plausible way for achieving a functional circadian clock, we adopted a transcriptional rewiring approach, artificially co-opting regulators of the circadian output pathways into the core-oscillator. Herein we describe one of these semi-synthetic clocks which maintains all basic circadian features but, notably, it also exhibits new attributes such as a "lights-on timer" logic, where clock phase is fixed at the end of the night. Our findings indicate that fundamental circadian properties such as period, phase and temperature compensation are differentially regulated by transcriptional and posttranslational aspects of the clockworks. - Eukaryotic circadian oscillators share a one-step transcription-translation negative-feedback loop (TTFL) circuit design, where a main transcriptional complex drives expression of a negative element that can inhibit its own expression. Here, a transcriptional rewiring strategy in the fungus N. crassa reveals that this basic circuit topology can be dramatically changed, while still yielding a functional semi-synthetic clock.Placing the transcription of the negative component frq under the control of the output promoter con-10 alters its regulation, while still yielding rhythmic expression. In the resulting functional semi-synthetic circuit, frq expression is now under the control of circadian clock downstream components. FRQ phosphorylation has a more pronounced effect on circadian period length and temperature compensation than its transcriptional regulation. In the semi-synthetic oscillator, the circadian rhythm phase is now determined by the moment the light is turned on. - Co-option of downstream transcription factors into the main transcriptional circuit of the circadian clock in the fungus Neurospora crassa leads to altered clock phase determination.