Journal of Molecular Biology
Minimum-Energy Path for a U6 RNA Conformational Change Involving Protonation, Base-Pair Rearrangement and Base Flipping
Introduction
U6 RNA is one of five snRNAs (U1, U2, U4, U5 and U6) that, along with more than 70 proteins, assemble on the pre-mRNA to form a large and dynamic complex called the spliceosome.1, 2 The spliceosome catalyzes precise excision of introns and ligation of the flanking exons through a two-step phosphotransesterification reaction resulting in production of mature mRNA. Once fully assembled, the spliceosome is activated through a number of structural rearrangements in the snRNAs, facilitated by protein components.3 These rearrangements result in disruption of the U4-U6 complex and formation of the U2-U6 complex (Fig. 1), which assists in splicing catalysis through substrate positioning and possibly chemistry.4 The association of U2 and U6 leads to formation of a highly conserved U6 internal stem–loop (U6 ISL).3
The secondary structure of the yeast U6 RNA was investigated by in vivo and in vitro dimethyl sulfate probing experiments, and these data are consistent with the presence of an asymmetric 1 × 2 internal loop in the center of the U6 ISL5 (Fig. 1). This highly conserved internal loop seems to be crucial for spliceosome function, since it coordinates an essential Mg2+ ion at the U80 position,6, 7 mutations inside the bulge sequence generate growth defects that are not fully suppressed by compensatory mutations in U4 RNA,8 and hydroxyl radical probing experiments place the human ISL internal loop in the vicinity of the 5′ splice site in activated spliceosomes.9 In addition, the internal-loop sequence found in the U6 ISL is also found in other RNAs. A database survey of 955 different RNA secondary structures found 12 occurrences of this 1 × 2 internal loop in 16S and 23S rRNAs and in RNase P.10
The structure and dynamics of the U6 ISL have been investigated by NMR spectroscopy.7, 11, 12, 13, 14, 15, 16 Consistent with the dimethyl sulfate probing results, the NMR solution structure determined at pH 7.0 shows that the U6 ISL contains a partially protonated C67–A79 wobble base pair and an unpaired U80 that is stacked in the helix.7, 11 In addition, comparison of the ISL structures at pH 7.0 and pH 5.7 indicates that the U80 nucleotide is in equilibrium between a stacked and a flipped-out conformation, with the latter predominating at low pH.12 The pH-dependent conformational switch appears to be induced by protonation of the A79 N1 atom (pKa = 6.5), which results in stabilization of the C67–A79 wobble pair and base-flipping of the neighboring U80 nucleotide.12 Analyses of NMR relaxation rates indicate that the lifetime of the protonated C67–A79 N1 base pair is 20 μs,12 while base-flipping of U80 occurs on the 80-μs time scale.13 Magnesium binding studies indicate that low pH inhibits metal ion binding, and the amount of metal ion binding is proportional to the fractional population of the stacked U80 (high pH) conformation.7, 13 Thus, the U6 ISL conformational equilibrium has the potential to regulate spliceosome activity.12, 13 However, the mechanism and associated energetics of this conformational transition have not been described, and, in particular, it is not clear why stacking of U80 is only favored upon disruption of the protonated C–A wobble pair. Although RNA is widely considered to be a dynamic molecule, the driving forces for structural rearrangements in RNA are poorly understood in general.
Since the population of protonated A79 is still significant at pH 7.0 (∼ 24%), we hypothesized that the pH 7.0 U6 ISL NMR structure is affected by conformational averaging due to interference from the low-pH conformation. If this hypothesis is true, then a high-pH structure should reveal a different conformation of the U6 ISL internal loop. Here we determine the NMR structure of U6 ISL at pH 8.0, and indeed this NMR structure reveals a different loop conformation with a C67–U80 pair and an unpaired A79. The previously determined low-pH (pH 5.7) structure and the pH 8.0 NMR structures were used as endpoints for computation of the minimum-energy path (MEP) for the conformational change using nudged elastic band (NEB) sampling.17 The results explain the physical basis for this conformational change, which involves a complex interplay of nucleobase protonation, alternative base-pair formation and base flipping.
Section snippets
NMR analysis
The U6 ISL RNA in this and previous studies incorporates an A62G substitution (Fig. 1), which has no effect on the growth rate of yeast at 30 °C5 and no effect on the overall structure of the ISL,7 but allows for optimal in vitro transcription using T7 RNA polymerase. Comparison of 2D 1H–1H nuclear Overhauser enhancement (NOE) spectroscopy (NOESY) spectra at pH 8.0 and pH 7.0 reveals no detectable difference in the NOE pattern (Fig. 2a). The high degree of similarity in the NMR spectra
Structures of the U6 RNA ISL
Analyses of NMR line shapes and relaxation rates indicated that internal-loop residues (C67, A79 and U80) of the U6 ISL display motion on both the picosecond-to-nanosecond and microsecond-to-millisecond time scales.12, 13 The U80 nucleotide adopts a stacked or looped-out conformation, depending on the protonation state of A79.7, 11, 12 It was previously hypothesized that an improved stacking free energy between A79+ and G81 was a driving force for the base-flipping transition.12
We have solved
RNA sample preparation
The U6 ISL RNA was prepared by in vitro transcription as previously described.7, 11 Sample conditions for NMR experiments were 1 mM RNA and 50 mM NaCl at pH 7.0 and 8.0. 2D TOCSY spectra were obtained in 50 mM NaCl and in 15 mM KCl, and no detectable differences were observed between the two ionic conditions. Partial alignment of 13C-labeled U6 ISL for RDC measurements was achieved by addition of Pf1 filamentous bacteriophage (17 mg/mL; ASLA Biotech, Ltd.) in deuterium oxide.
NMR spectroscopy
All spectra were
Acknowledgements
The authors thank Professors David A. Brow, David H. Mathews, Brent Znosko and Qiang Cui for helpful comments and suggestions. This study made use of the National Magnetic Resonance Facility at Madison, which is supported by National Institutes of Health (NIH) grants P41RR02301 (BRTP/NCRR) and P41GM66326 (NIGMS). Additional equipment was purchased with funds from the University of Wisconsin, the NIH (RR02781, RR08438), the National Science Foundation (DMB-8415048, OIA-9977486, BIR-9214394), and
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