Key Concepts:

Figure 1. Competition of circadian strains in different LD cycles. (a) Different strains of S. elongatus were mixed together in batch cultures and grown in competition under different LD cycles. Every 8 days, the cultures were diluted with fresh medium. At various times during the competition, aliquots were plated as single colonies and their luminescence rhythms were monitored to determine the frequency distribution of the different circadian phenotypes. (b) In competitions between wild-type and arrhythmic strains, the arrhythmic strain is rapidly out-competed in LD cycles, but slowly defeats wild-type in constant light (LL, nonselective conditions). (c) In competitions among strains that are rhythmic, the strain whose endogenous free-running period (FRP) most closely matched that of the environmental LD cycle was able to out-compete strains with a nonoptimal FRP. In LL, all the strains were able to maintain their initial fraction in the population. (Data are smoothed curves based on raw data from: (b) Woelfle et al., 2004; (c) Ouyang et al., 1998.)

Figure 2. Rhythms of KaiC proteins in vivo and in vitro. (a) Rhythmic KaiC phosphorylation in vivo at different times in constant light (LL). Samples were collected every 4 h in constant light and processed for SDS PAGE and immunoblotting. The lowest band is hypo-phosphorylated KaiC and the upper bands are various forms of phosphorylated KaiC. Hyper-phosphosphorylated KaiC is most prevalent at 8–16 and 32–40 h. (b) Rhythmic KaiC phosphorylation in the in vitro reaction. Purified KaiA, KaiB and KaiC are combined with ATP in vitro and samples collected every 3 h and processed for SDS PAGE and staining. Four bands are obvious in these samples: hypo-phosphorylated KaiC and KaiC phosphorylated at the S431, T432 or S431/T432 residues (see labels on the right side of the panel). (c) Rhythms of KaiA/B/C complexes during the in vitro rhythm as visualised by electron microscopy of the protein mixtures. The rhythm of KaiC phosphorylation in vitro is shown by the solid line, whereas the pie charts show the proportions of the various complexes. For example, only free KaiC & KaiA·KaiC complexes are present during the rising phase of KaiC phosphorylation, whereas free KaiC, KaiA·KaiC, KaiB·KaiC and KaiA·KaiB·KaiC are all present during the dephosphorylation phase. (d) KaiA/KaiB/KaiC complexes themselves are shown with colour coding that corresponds with the pie charts depicted in (c) (adapted from Johnson et al., 2008 [Current Biology]. Copyright Elsevier).

Figure 3. Models for the in vitro and in vivo oscillators. (a) The diagram represents a model for the phosphorylation cycle of a KaiC hexamer and its association with KaiA and KaiB (Mori et al., 2007). A KaiC monomer is shown as a double circle that can form a hexamer. KaiC hexamers can associate/dissociate with KaiA and/or KaiB. KaiC hexamers are depicted in two conformational states: a default KaiC status (light blue colour=autokinase) and an altered KaiC state that has undergone a conformational change (darker blue colour). Red dots are phosphates attached to the S431 and T432 phosphorylation sites on KaiC. Starting from a hypo-phosphorylated state (), rapid binding and unbinding of KaiA facilitates autophosphorylation until the KaiC hexamers are hyper-phosphorylated (state ). In this model, KaiB is assumed to preferentially associate with hyper-phosphorylated KaiC; there is a simultaneous conformational change of KaiC to a new state (KaiC depicted as a darker blue hexamer=autophosphatase). The KaiC hexamer de-phosphorylates through states and until it is no longer phosphorylated, at which time it reverts to the original conformation (state ). Robustness is maintained by synchronization of KaiC hexameric status via monomer exchange (Mori et al., 2007; Ito et al., 2007), depicted by ‘dumbbell’ KaiC monomers exchanging with KaiC hexamers in the central region of the figure. The rate of this exchange is highest during the dephosphorylation phase (Ito et al., 2007), as indicated by the thicker exchange arrow in this phase. (b) In vivo, a self-sustained post-translational oscillator (PTO) is embedded within a transcription and translation feedback loop (TTFL). The PTO is shown in greater detail in (a). New synthesis of KaiC monomers feeds into the KaiABC oscillator as non-phosphorylated hexamers or monomers that exchange into pre-existing hexamers. Some configuration of KaiC (here shown as the dephosphorylating phase, but it could be any phase of the PTO) mediates rhythmic DNA torsion/compaction and transcriptional factor activity to control global transcription of all promoters, including those driving expression of the essential clock genes kaiA, kaiB and kaiC. TFs, transcriptional factors. See Johnson et al. (2008) and Qin et al. (2010).

Figure 4. Structures of the key clock proteins KaiA, KaiB and KaiC. (a) Side view of the KaiC hexamer, showing the CI and CII domains. The six different subunits are illustrated in different colours (from Pattanayek et al., 2004). (b) The pore of KaiC viewed from the CI opening. In (a) and (b), the ‘ball-and-stick’ molecules are ATP. (c) The three essential residues in the CII domain for autophosphorylation: T426, S431 and T432 (from Xu et al., 2004). (d) The KaiA dimer, each subunit is in a different colour, showing the ‘domain swapping’ characteristic of KaiA (from Ye et al., 2004). (e) The KaiB tetramer, each subunit is in a different colour (from Hitomi et al., 2005).