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Dynamics in the U6 RNA intramolecular stem-loop: a base flipping conformational change
Nicholas J Reiter1, Heike Blad, Frits Abildgaard
1Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.
Biochemistry
|October 27, 2004
Summary
The U6 RNA stem-loop structure changes conformation at lower pH, flipping out a nucleotide. This base flipping is crucial for metal ion binding in the spliceosome and group II introns.
Area of Science:
- Molecular Biology
- Structural Biology
- RNA Biology
Background:
- The U6 RNA intramolecular stem-loop (ISL) is vital for spliceosome function, binding essential metal ions for pre-messenger RNA splicing.
- A key feature is the metal-binding internal loop containing a C67-(+)A79 base pair and a U80 nucleotide.
Purpose of the Study:
- To investigate the structural dynamics of the U6 ISL at different pH levels.
- To understand the mechanism and implications of pH-induced conformational changes, specifically base flipping.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy to determine solution structures.
- Analysis of residual dipolar couplings to assess conformational dynamics.
- Protonation exchange lifetime measurements.
Main Results:
- Protonation of the C67-(+)A79 base pair occurs with a lifetime of approximately 20 microseconds.
- At pH 5.7, compared to pH 7.0, the U80 nucleotide is flipped out into the major groove.
- This base flipping involves significant conformational rearrangements, including sugar pucker inversion and backbone rearrangement, altering helix alignment.
Conclusions:
- pH-dependent conformational changes in the U6 ISL, particularly U80 base flipping, are critical for its function.
- These structural alterations reveal conserved mechanisms between the spliceosome and self-splicing group II introns.
- Base flipping at metal-binding sites appears to be a conserved feature in RNA splicing machinery.