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.

NMR studies of dynamics in RNA and DNA by 13C relaxation

“…It was shown that the U6 intramolecular stem loop from the U6 spliceosomal snRNA has a pH dependent conformational equilibrium that involved helical movement of *258 and base flipping of U80. 81 A recent 13 C T 1 power dependence analysis of this system revealed the presence of a global conformational transition at a rate of about 84 s, that was consistent with the conversion between pH dependent states. 67 Concerning DNA, to date only the terminal residues have displayed flexibility among the oligonucleotides that have been studied.…”

pH-dependent structural changes of helix 69 from Escherichia coli 23S ribosomal RNA

“…The overall thermodynamic stability and/or structural properties of some RNAs are influenced by pH (Durant and Davis 1999;Nixon and Giedroc 2000;Bayfield et al 2004;Reiter et al 2004). For example, formation of a protonated A + dC base pair in the anticodon stem-loop of tRNA Lys,3 stabilizes the hairpin structure (Durant and Davis 1999).…”

Alternative Roles for Metal Ions in Enzyme Catalysis and the Implications for Ribozyme Chemistry

“…Nevertheless, many cases of shifted acidities and basicities of nucleobases in nucleic acid structures are known. [154][155][156][157][158][159][160][161][162][163][164][165] One way to strongly influence pK a values, the geometry, and other fundamental properties of nucleotides are bound metal ions and other fixed charges. 66,75,102,153 In particular, the effects of metal ions on acidity of nucleotide functional groups has been quantitatively investigated in nucleobase model systems.…”

“…One of the most common types of pK a shift found in complex RNA structures involves the N1 moiety of protonated A + ·C base pairs. [154][155][156][157][158][159][160][161][162][163] There are two such A + ·C pairs in the extended U6 internal stem loop of spliceosomal i.e. raised by more than 2 log units.…”

“…raised by more than 2 log units. 158,159 A second example is observed in the VS ribozyme, where a corresponding A + ·C pair is located one base pair downstream from the cleavage site. 160,161 In both the spliceosome and the VS ribozyme, these positively charged A + ·C base pairs are likely to play an important role in catalysis.…”