Dynamics in the U6 RNA Intramolecular Stem-Loop: A Base Flipping Conformational Change
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- 作者：Nicholas J. Reiter ; Heike Blad ; Frits Abildgaard ; and Samuel E. Butcher
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：November 2, 2004
- 年：2004
- 卷：43
- 期：43
- 页码：13739 - 13747
- 全文大小：615K
- 年卷期：v.43,no.43(November 2, 2004)
- ISSN：1520-4995
文摘
The U6 RNA intramolecular stem-loop (ISL) structure is an essential component of thespliceosome and binds a metal ion required for pre-messenger RNA splicing. The metal binding internalloop region of the stem contains a partially protonated C67-+A79 base pair (pKa = 6.5) and an unpairedU80 nucleotide that is stacked within the helix at pH 7.0. Here, we determine that protonation occurswith an exchange lifetime of approximately 20 
s and report the solution structures of the U6 ISL at pH5.7. The differences between pH 5.7 and 7.0 structures reveal that the pH change significantly alters theRNA conformation. At lower pH, U80 is flipped out into the major groove. Base flipping involves apurine 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 pHindicates that base flipping is not restricted to a local conformational change. Rather, base flipping altersthe alignment of the upper and lower helices. The alternative conformations of the U6 ISL reveal strikingstructural similarities with both the NMR and crystal structures of domain 5 of self-splicing group IIintrons. These structures suggest that base flipping at an essential metal binding site is a conserved featureof the splicing machinery for both the spliceosome and group II self-splicing introns.
s and report the solution structures of the U6 ISL at pH5.7. The differences between pH 5.7 and 7.0 structures reveal that the pH change significantly alters theRNA conformation. At lower pH, U80 is flipped out into the major groove. Base flipping involves apurine 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 pHindicates that base flipping is not restricted to a local conformational change. Rather, base flipping altersthe alignment of the upper and lower helices. The alternative conformations of the U6 ISL reveal strikingstructural similarities with both the NMR and crystal structures of domain 5 of self-splicing group IIintrons. These structures suggest that base flipping at an essential metal binding site is a conserved featureof the splicing machinery for both the spliceosome and group II self-splicing introns.