PDBsum entry 2kf0

RNA

PDB id

2kf0

Contents

			DNA/RNA

References listed in PDB file

Key reference

Title

Minimum-Energy path for a u6 RNA conformational change involving protonation, Base-Pair rearrangement and base flipping.

Authors

V.Venditti, L.Clos, N.Niccolai, S.E.Butcher.

Ref.

J Mol Biol, 2009, 391, 894-905. [DOI no: 10.1016/j.jmb.2009.07.003]

PubMed id

19591840

Abstract

The U6 RNA internal stem-loop (U6 ISL) is a highly conserved domain of the spliceosome that is important for pre-mRNA splicing. The U6 ISL contains an internal loop that is in equilibrium between two conformations controlled by the protonation state of an adenine (pK(a)=6.5). Lower pH favors formation of a protonated C-A(+) wobble pair and base flipping of the adjacent uracil. Higher pH favors stacking of the uracil and allows an essential metal ion to bind at this position. Here, we define the minimal-energy path for this conformational transition. To do this, we solved the U6 ISL structure at higher pH (8.0) in order to eliminate interference from the low-pH conformer. This structure reveals disruption of the protonated C-A(+) pair and formation of a new C-U pair, which explains the preference for a stacked uracil at higher pH. Next, we used nudged elastic band molecular dynamics simulations to calculate the minimum-energy path between the two conformations. Our results indicate that the C-U pair is dynamic, which allows formation of the more stable C-A(+) pair upon adenine protonation. After formation of the C-A(+) pair, the unpaired uracil follows a minor-groove base-flipping pathway. Molecular dynamics simulations suggest that the extrahelical uracil is stabilized by contacts with the adjacent helix.



	Figure 1. Fig. 1. (a) Schematic diagram of Saccharomyces cerevisiae U6 snRNA and its conformations in the free U6 snRNP, the U4-U6 di-snRNP, and the U2–U6 snRNA complex in the activated spliceosome. The U6 ISL is indicated with a dashed box. (b) Sequence and secondary structure of the U6 ISL used in this study. The previously described A62G substitution that does not affect the overall structure of the U6 ISL is indicated with a box. The secondary structure is shown in the low-pH form, with a protonated A79 (indicated with a +) and a bulged U80.		Figure 5. Fig. 5. Endpoint structures used for determining the MEP. (a) Stacked conformation (pH 8.0 structure). (b) Energy-minimized U80 flipped-out conformation. Both structures are shown with A79 protonated.