title: Dynamics in the U6 RNA intramolecular stem-loop: a base flipping conformational change.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Reiter
authors.forename: Heike
authors.initials: H
authors.lastname: Blad
authors.forename: Frits
authors.initials: F
authors.lastname: Abildgaard
authors.forename: Samuel E
authors.initials: SE
authors.lastname: Butcher
mesh_terms: D020029:Base Pairing; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D011522:Protons; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics; D012342:RNA, Small Nuclear / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D011522:Protons; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA