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Title
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Dynamics in the U6 RNA intramolecular stem-loop: a base flipping conformational change.
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Authors
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N.J.Reiter,
H.Blad,
F.Abildgaard,
S.E.Butcher.
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Ref.
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Biochemistry, 2004,
43,
13739-13747.
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PubMed id
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Abstract
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The U6 RNA intramolecular stem-loop (ISL) structure is an essential component of
the spliceosome and binds a metal ion required for pre-messenger RNA splicing.
The metal binding internal loop region of the stem contains a partially
protonated C67-(+)A79 base pair (pK(a) = 6.5) and an unpaired U80 nucleotide
that is stacked within the helix at pH 7.0. Here, we determine that protonation
occurs with an exchange lifetime of approximately 20 micros and report the
solution structures of the U6 ISL at pH 5.7. The differences between pH 5.7 and
7.0 structures reveal that the pH change significantly alters the RNA
conformation. At lower pH, U80 is flipped out into the major groove. Base
flipping involves a purine stacking interaction of flanking nucleotides,
inversion of the sugar pucker 5' to the flipped base, and phosphodiester
backbone rearrangement. Analysis of residual dipolar couplings as a function of
pH indicates that base flipping is not restricted to a local conformational
change. Rather, base flipping alters the alignment of the upper and lower
helices. The alternative conformations of the U6 ISL reveal striking structural
similarities with both the NMR and crystal structures of domain 5 of
self-splicing group II introns. These structures suggest that base flipping at
an essential metal binding site is a conserved feature of the splicing machinery
for both the spliceosome and group II self-splicing introns.
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