Single-molecule fluorescence resonance energy transfer assays reveal heterogeneous folding ensembles in a simple RNA stem-loop.
We have examined the folding ensembles present in solution for a series of RNA oligonucleotides that encompass the replicase translational operator stem-loop of the RNA bacteriophage MS2. Single-molecule (SM) fluorescence assays suggest that these RNAs exist in solution as ensembles of differentially base-paired/base-stacked states at equilibrium. There are two distinct ensembles for the wild-type sequence, implying the existence of a significant free energy barrier between "folded" and "unfolded" ensembles. Experiments with sequence variants are consistent with an unfolding mechanism in which interruptions to base-paired duplexes, in this example by the single-stranded loop and a single-base bulge in the base-paired stem, as well as the free ends, act as nucleation points for unfolding. The switch between folded and unfolded ensembles is consistent with a transition that occurs when all base-pairing and/or base-stacking interactions that would orientate the legs of the RNA stem are broken. Strikingly, a U-to-C replacement of a residue in the loop, which creates a high-affinity form of the operator for coat protein binding, results in dramatically different (un)folding behaviour, revealing distinct subpopulations that are either stabilised or destabilised with respect to the wild-type sequence. This result suggests additional reasons for selection against the C-variant stem-loop in vivo and provides an explanation for the increased affinity.
Gell
Christopher
C
School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, UK; Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS29JT, UK.
Sabir
Tara
T
Westwood
Jonathan
J
Rashid
Abdul
A
Smith
D Alastair M
DA
Harris
Sarah-Anne
SA
Stockley
Peter G
PG
D001483:Base Sequence; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D017909:Levivirus / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013057:Spectrum Analysis; D013816:Thermodynamics
D036022:Capsid Proteins; D012367:RNA, Viral