Kim, Yong-Ick (2008-12). The day/night switch of the circadian clock of synechococcus elongatus and hydrogen bonds of dna and rna. Doctoral Dissertation. | Thesis individual record

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, 2h?13C2, are sensitive to the hydrogen-bond
distance between the N1 of adenine and the N3 of uracil or thymine, which supports the
notion that 2h?13C2 is sensitive to hydrogen-bond strength. Calculated 2h?13C2 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 2h?13C2 values. Thus, we
show experimentally and computationally that the C7 methyl group of thymine has no
measurable affect on 2h?13C2 values. Furthermore, 2h?13C2 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 2h?13C2 is context dependent and that this dependence is similar for
RNA and DNA.

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