O'Shea Lab > People > Joe Markson
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| The Mechanism
of an in vitro, Protein-based Circadian
Oscillator |
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Circadian clocks coordinate the physiological
state of an organism with the diurnal day-night
cycle. These clocks traditionally have been viewed
as transcription-translation oscillators (TTOs),
in which delayed negative transcriptional feedback
drives 24-hour oscillations in the concentration
of a central protein called the “state variable.” The
TTO model originally was applied even to the circadian
clock of cyanobacteria, the simplest organisms
with known circadian rhythms. The cyanobacterial
clock centers around three proteins called KaiA,
KaiB, and KaiC; the phosphorylation state of the
hexameric autokinase/autophosphatase KaiC is the
state variable, with KaiA and KaiB modulating its
enzymatic activity. However, Nakajima et al reported
last year the astonishing result that the clock
can be reconstituted in vitro with just the three
Kai proteins and ATP. In this in vitro clock, the
phosphorylation state of KaiC oscillates with a
24-hour, temperature-compensated period, just as
it does in vivo. Kageyama et al recently reported
some information regarding stoichiometries, rates,
and concentration dependence in the Kai system,
but a detailed understanding remains elusive.
We recently began studying the in vitro cyanobacterial
clock in order to obtain a mechanistic understanding
of this extraordinary system. Our first experiments
will allow us to discriminate among different classes
of models. We will conduct quantitative studies
of the time-varying stoichiometries of the KaiA/B/C
complex, the phosphorylation state of the KaiC
hexamer, and other fundamental properties of the
system. We also will examine the variation in these
properties within the protein population at any
given time. All along, we will integrate our experimental
findings with mathematical simulations. We hope
to be able to explain how this three-component
oscillator cycles robustly with so few components
in the face of intrinsically noisy chemical reactions
and even variations in concentration and temperature.
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