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Description:
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The circadian oscillator of the cyanobacterium Synechococcus elongatus is
composed of only three proteins , KaiA , KaiB , and KaiC , which together with ATP can
generate a self -sustained ~24 hour oscillation of KaiC phosphorylation for several days .
KaiA induces KaiC to autophosphorylate whereas KaiB blocks the stimulation of KaiC
by KaiA , which allows KaiC to autodephosphorylate . We propose and support a model
in which the C -terminal loops of KaiC , the “A -loops” , are the master switch that
determines overall KaiC activity . When the A -loops are in their buried state , KaiC is an
autophosphatase . When the A -loops are exposed , however , KaiC is an autokinase . The
data suggest that KaiA stabilizes the exposed state of the A -loops through direct binding .
We also show evidence that if KaiA cannot stabilize the exposed state KaiC remains
hypophosphorylated . We propose that KaiB inactivates KaiA by preventing it from
stabilizing the exposed state of the A -loops . Thus , KaiA and KaiB likely act by shifting
the dynamic equilibrium of the A -loops between exposed and buried states , which shifts
the balance of autokinase and autophosphatase activities of KaiC . A -loop exposure likely moves the ATP closer to the sites of phosphorylation and we show evidence in
support of how this movement may be accomplished .
Density functional theory calculations of isolated Watson–Crick A :U and A :T
base pairs predict that adenine 13C2 trans -hydrogen bond deuterium isotope shifts due to
isotopic substitution at the pyrimidine H3 , 2hΔ13C2 , are sensitive to the hydrogen -bond
distance between the N1 of adenine and the N3 of uracil or thymine , which supports the
notion that 2hΔ13C2 is sensitive to hydrogen -bond strength . Calculated 2hΔ13C2 values at
a given N1–N3 distance are the same for isolated A :U and A :T base pairs . Replacing
uridine residues in RNA with 5 -methyl uridine and substituting deoxythymidines in
DNA with deoxyuridines do not statistically shift empirical 2hΔ13C2 values . Thus , we
show experimentally and computationally that the C7 methyl group of thymine has no
measurable affect on 2hΔ13C2 values . Furthermore , 2hΔ13C2 values of modified and
unmodified RNA are more negative than those of modified and unmodified DNA , which
supports our hypothesis that RNA hydrogen bonds are stronger than those of DNA . It is
also shown here that 2hΔ13C2 is context dependent and that this dependence is similar for
RNA and DNA . |