Complex dynamics under tension in a high-efficiency frameshift stimulatory structure

Abstract

Specific structures in mRNA can stimulate programmed ribosomal frameshifting (PRF). PRF efficiency can vary enormously between different stimulatory structures, but the features that lead to efficient PRF stimulation remain uncertain. To address this question, we studied the structural dynamics of the frameshift signal from West Nile virus (WNV), which stimulates −1 PRF at very high levels and has been proposed to form several different structures, including mutually incompatible pseudoknots and a double hairpin. Using optical tweezers to apply tension to single mRNA molecules, mimicking the tension applied by the ribosome during PRF, we found that the WNV frameshift signal formed an unusually large number of different metastable structures, including all of those previously proposed. From force-extension curve measurements, we mapped 2 mutually exclusive pathways for the folding, each encompassing multiple intermediates. We identified the intermediates in each pathway from length changes and the effects of antisense oligomers blocking formation of specific contacts. Intriguingly, the number of transitions between the different conformers of the WNV frameshift signal was maximal in the range of forces applied by the ribosome during −1 PRF. Furthermore, the occupancy of the pseudoknotted conformations was far too low for static pseudoknots to account for the high levels of −1 PRF. These results support the hypothesis that conformational heterogeneity plays a key role in frameshifting and suggest that transitions between different conformers under tension are linked to efficient PRF stimulation.programmed ribosomal frameshifting | RNA folding | pseudoknots | force spectroscopy | West Nile virus

Modeling the structure of the frameshift stimulatory pseudoknot in SARS-CoV-2 reveals multiple possible conformers

“…This difference can be explained by the fact that only a single initial structure was explored in that work: the three fold topologies we observe are sufficiently different that they cannot interconvert without substantial unfolding of the S1/S2 region, and they are each sufficiently stable that such unfolding is very unlikely (as seen in our simulations). Furthermore, the existence of multiple structures has been seen previously in various frameshift signals (26)(27)(28). Indeed, it is entirely consistent with the hypothesis (29-31) that high-efficiency stimulatory structures such as that from SARS-CoV-2which induces −1 PRF at a rate of ~20-30% (6)-have high conformational heterogeneity and hence forms more than one structure.…”

“…X-ray scattering profiles can be predicted from these models and used to analyze small-and wide-angle x-ray scattering measurements, to confirm which (if any) of these conformations are populated and in what kind of mixture (33,34). The models could also be compared to single-molecule measurements of pseudoknot folding, which could detect heterogeneous populations of different conformers and characterize the sequence of intermediate states formed during the folding of each one (28,35,36). These models should also prove useful for drug discovery efforts, facilitating structure-based searches for compounds that attenuate the virus by altering −1 PRF.…”

Mechanically stable knot formed by strand threading in Zika virus RNA confers RNase resistance

“…Fitting the FECs before and after the low-force rip to extensible worm-like chain (WLC) models ( Fig 1C, dotted lines) (17), we found a contour-length change of ΔLc = 6.1 ± 0.4 nm, only a small fraction of the 38.3 nm expected for complete unfolding (14). The xrRNA thus remained in a mostly folded intermediate, denoted Ir, even at 60 pN-well above the unfolding forces reported for any other RNA structures (18)(19)(20)(21)(22)(23)(24) and in the same range as the duplex overstretching transition (25). Indeed, in most of these curves, unfolding the xrRNA completely required holding it at the overstretching force for at least 2 s; as a result, Ir unfolding occurred at the same time as handle overstretching and was difficult to distinguish (Fig.…”

“…Unfolding and refolding of secondary structures alone, without any tertiary structures, typically occurs at forces in the range 10-25 pN, relatively close to equilibrium so that unfolding and refolding forces are similar (26). In contrast, tertiary structures typically unfold at forces > 25 pN, and usually display a wide distribution of unfolding forces and strong hysteresis when refolding (18)(19)(20)(21)(22). Only structural changes that alter the end-to-end length of the RNA can be observed directly by SMFS; conformational changes that leave the end-to-end length unchanged (e.g.…”

“…1D, Fig. S1C), in the 10-20 pN range characteristic of secondary structures (26), followed by a final transition at higher force, in the ~30-50 pN range characteristic for unfolding tertiary structures (18)(19)(20)(21)(22). The total ΔLc for complete unfolding in these FECs, 36 ± 1 nm, was slightly shorter than for the native fold, indicating that the xrRNA started in a state close to the native fold.…”