Revealing a Two-Loop Transcriptional Feedback Mechanism in the Cyanobacterial Circadian Clock

Stefanie Hertel,Christian Brettschneider,Ilka M Axmann,Denis Thieffry

doi:10.1371/journal.pcbi.1002966

Stefanie Hertel, Christian Brettschneider + Show 2 more

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https://doi.org/10.1371/journal.pcbi.1002966

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Abstract

Molecular genetic studies in the circadian model organism Synechococcus have revealed that the KaiC protein, the central component of the circadian clock in cyanobacteria, is involved in activation and repression of its own gene transcription. During 24 hours, KaiC hexamers run through different phospho-states during daytime. So far, it has remained unclear which phospho-state of KaiC promotes kaiBC expression and which opposes transcriptional activation. We systematically analyzed various combinations of positive and negative transcriptional feedback regulation by introducing a combined TTFL/PTO model consisting of our previous post-translational oscillator that considers all four phospho-states of KaiC and a transcriptional/translational feedback loop. Only a particular two-loop feedback mechanism out of 32 we have extensively tested is able to reproduce existing experimental observations, including the effects of knockout or overexpression of kai genes. Here, threonine and double phosphorylated KaiC hexamers activate and unphosphorylated KaiC hexamers suppress kaiBC transcription. Our model simulations suggest that the peak expression ratio of the positive and the negative component of kaiBC expression is the main factor for how the different two-loop feedback models respond to removal or to overexpression of kai genes. We discuss parallels between our proposed TTFL/PTO model and two-loop feedback structures found in the mammalian clock.

Highlights

Photoautotrophic organisms like plants and cyanobacteria are subjected to a daily light-dark rhythm and have been demonstrated to possess a self-sustained circadian clock
We investigated the impact of each KaiC phospho-state on kaiBC expression by introducing a model that combines the circadian transcription/translation rhythm with the KaiABC-protein oscillator
It turns out that the kaiBC expression and KaiC phosphorylation dynamics in wild type and kai mutants can only be described by one mechanism: threonine and double phosphorylated KaiC hexamers activate kaiBC expression and the unphosphorylated state suppresses it

Summary

Introduction

Photoautotrophic organisms like plants and cyanobacteria are subjected to a daily light-dark rhythm and have been demonstrated to possess a self-sustained circadian clock. The simplest circadian clock ticks in cyanobacteria. It consists of just three proteins KaiA, KaiB and KaiC composing a post-translational oscillator (PTO). This unique three-protein clock is well described for Synechococcus elongatus PCC 7942 (hereafter Synechococcus). ATPase and kinase/phosphatase occur in the C1 and C2 rings of the KaiC hexamer, respectively. The consensus view is that the ATPase crosstalks with the kinase/phosphatase through a structural coupling between the two rings [3]. KaiC forms hexamers and each KaiC monomer within the hexamer possesses two main phosphorylation sites (T432 and S431) [6]. The four forms of KaiC cycle in a stepwise fashion: unphosphorylated (U-KaiC), threonine phosphorylated (TKaiC), both residues phosphorylated (D-KaiC), and serine phosphorylated (S-KaiC) [7,8]

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