Single-Molecule Measurements of the CCR5 mRNA Unfolding Pathways

Abstract

Secondary or tertiary structure in an mRNA, such as a pseudoknot, can create a physical barrier that requires the ribosome to generate additional force to translocate. The presence of such a barrier can dramatically increase the probability that the ribosome will shift into an alternate reading frame, in which a different set of codons is recognized. The detailed biophysical mechanism by which frameshifting is induced remains unknown. Here we employ optical trapping techniques to investigate the structure of a -1 programmed ribosomal frameshift (-1 PRF) sequence element located in the CCR5 mRNA, which encodes a coreceptor for HIV-1 and is, to our knowledge, the first known human -1 PRF signal of nonviral origin. We begin by presenting a set of computationally predicted structures that include pseudoknots. We then employ what we believe to be new analytical techniques for measuring the effective free energy landscapes of biomolecules. We find that the -1 PRF element manifests several distinct unfolding pathways when subject to end-to-end force, one of which is consistent with a proposed pseudoknot conformation, and another of which we have identified as a folding intermediate. The dynamic ensemble of conformations that CCR5 mRNA exhibits in the single-molecule experiments may be a significant feature of the frameshifting mechanism.

Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for −1 ribosomal frameshifting stimulation

“…After the formation of the pseudoknot structure at low forces, the extension versus time (constant-force) trajectory is stable without hopping (Fig. 4d), which is consistent with the fact that pseudoknots have relatively high unfolding forces as previously reported37404849. We observed, based on the extension versus time curves (see Supplementary Fig.…”

“…An H-type pseudoknot structure typically unfolds cooperatively at a relatively high force compared to a hairpin37404849. In our force-ramp experiments, all sequences refolded into pseudoknot structures as indicated by the subsequent unfolding traces with a relatively high unfolding force (Fig.…”

“…However, the molecular determinants of the downstream mRNA structure stimulating −1 frameshifting efficiency are still unclear. Single-molecule mechanical pulling studies using optical tweezers have provided insight into the possible correlation between frameshifting stimulatory mRNA structures and −1 frameshifting efficiency353637383940. Two studies showed a correlation between the frameshifting efficiency and the mechanical strength of an mRNA pseudoknot3537.…”

Probing the structural dynamics of proteins and nucleic acids with optical tweezers

“…Early work identifying a correlation between the pseudoknot unfolding force and À1 PRF stimulation efficiency [63,64] was shown not to extend to a larger panel of viral pseudoknots; instead, À1 PRF efficiency was correlated with pseudoknot conformational plasticity [65 ], reflected in the tendency of the RNA to refold into alternative structures (Figure 2c,d). The importance of pseudoknot dynamics in stimulating frameshifting was corroborated by studies of the frameshift signal from human CCR5 mRNA, showing that it manifests several distinct unfolding pathways when mechanically destabilized [66], and by measurements of the effect of a ligand that abolishes À1 PRF upon binding specifically to the pseudoknot from the SARS coronavirus [67], showing that the ligand reduced the conformational plasticity of the pseudoknot proportional to the amount of ligand bound (Figure 2c inset) [68]. While such studies provide important insights, they investigate only one part of the À1 PRF mechanism, which involves interactions between many different elements.…”

Reconstructing Folding Energy Landscape Profiles from Nonequilibrium Pulling Curves with an Inverse Weierstrass Integral Transform

“…Reconstructing quantitative energy landscapes should therefore provide insight into how the dynamics and energetics of such RNA structures relate to their biological function, possibly aiding the development of therapeutics. The mechanical unfolding of pseudoknots has been studied previously with SMFS [26][27][28][29][30][31], but the full profile of the energy landscape of a pseudoknot has not yet been experimentally reconstructed.…”