The simple circadian oscillator found in cyanobacteria can be reconstituted in vitro using three proteins-KaiA, KaiB, and KaiC. The total phosphorylation level of KaiC oscillates with a circadian period, but the mechanism underlying its sustained oscillation remains unclear. We have shown that four forms of KaiC differing in their phosphorylation state appear in an ordered pattern arising from the intrinsic autokinase and autophosphatase rates of KaiC and their modulation by KaiA. Kinetic and biochemical data indicate that one of these phosphoforms inhibits the activity of KaiA via interaction with KaiB, providing the crucial feedback that sustains oscillation. A mathematical model constrained by experimental data quantitatively reproduces the circadian period and the distinctive dynamics of the four phosphoforms.Circadian clocks coordinate metabolism and behavior with diurnal cycles in the environment (1). These clocks traditionally have been understood as transcriptional feedback oscillators in which clock genes repress their own synthesis, creating negative feedback that drives oscillation (1). However, pioneering work by Kondo and colleagues has shown that the circadian clock of the cyanobacterium Synechococcus elongatus requires neither transcription nor translation (2), and circadian oscillations can be reconstituted in vitro using only three proteins: KaiA, KaiB, and KaiC (3).KaiC is a hexameric enzyme (4) that can autophosphorylate (5) and autodephosphorylate (6) at both serine 431 (S431) and threonine 432 (T432) (7,8). The dimeric KaiA (9, 10) enhances the autophosphorylation of KaiC (11), whereas KaiB antagonizes the activity of . In the absence of KaiA, KaiC fully dephosphorylates (9). Complexes form between the Kai proteins (14), and the relative proportions of the KaiC-containing complexes oscillate (9, 15).The amount of phosphorylated KaiC oscillates with a circadian period (11). However, the total level of phosphorylation cannot be the only dynamical variable controlling the oscillator since it traverses the same value twice each day, but each time in a different † To whom correspondence should be addressed. E-mail: erin_oshea@harvard.edu. * These authors contributed equally to this work. HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript direction (increasing during the subjective day and decreasing during the subjective night). Previous mathematical models have treated both phosphorylation sites as functionally equivalent (16)(17)(18)(19)(20)(21)(22)(23)(24), and have proposed additional dynamical variables arising from persistent conformational changes (18,(20)(21)(22)(23)(24) or long-lived heterocomplexes (16, 17); we hypothesized that additional variables could be found by examining the pattern of multisite phosphorylation of KaiC during the circadian cycle.We measured the time dependence of phosphorylation at S431 and T432 by SDS-PAGE (Fig. 1, A and B) and mass spectrometry (Fig. 1B), quantifying the four possible phosphorylation states: unphosphorylated (U-KaiC), p...

The cyanobacterial circadian clock generates genome-wide transcriptional oscillations and regulates cell division, but the underlying mechanisms are not well understood. Here, we show that the response regulator RpaA serves as the master regulator of these clock outputs. Deletion of rpaA abrogates gene expression rhythms globally and arrests cells in a dawn-like expression state. Although rpaA deletion causes core oscillator failure by perturbing clock gene expression, rescuing oscillator function does not restore global expression rhythms. We show that phosphorylated RpaA regulates the expression of not only clock components, generating feedback on the core oscillator, but also a small set of circadian effectors that, in turn, orchestrate genome-wide transcriptional rhythms. Expression of constitutively active RpaA is sufficient to switch cells from a dawn-like to a dusk-like expression state as well as to block cell division. Hence, complex global circadian phenotypes can be generated by controlling the phosphorylation of a single transcription factor.