Characterization of the Mechanical Unfolding of RNA Pseudoknots

Abstract

The pseudoknot is an important RNA structural element that provides an excellent model system for studying the contributions of tertiary interactions to RNA stability and to folding kinetics. RNA pseudoknots are also of interest because of their key role in the control of ribosomal frameshifting by viral RNAs. Their mechanical properties are directly relevant to their unfolding by ribosomes during translation. We have used optical tweezers to study the kinetics and thermodynamics of mechanical unfolding and refolding of single RNA molecules. Here we describe the unfolding of the frameshifting pseudoknot from infectious bronchitis virus (IBV), three constituent hairpins, and three mutants of the IBV pseudoknot. All four pseudoknots cause -1 programmed ribosomal frameshifting. We have measured the free energies and rates of mechanical unfolding and refolding of the four frameshifting pseudoknots. Our results show that the IBV pseudoknot requires a higher force than its corresponding hairpins to unfold. Furthermore, its rate of unfolding changes little with increasing force, in contrast with the rate of hairpin unfolding. The presence of Mg(2+) significantly increases the kinetic barriers to unfolding the IBV pseudoknot, but has only a minor effect on the hairpin unfolding. The greater mechanical stability of pseudoknots compared to hairpins, and their kinetic insensitivity to force supports the hypothesis that -1 frameshifting depends on the difficulty of unfolding the mRNA.

Mechanical Unfolding of the Beet Western Yellow Virus −1 Frameshift Signal

“…Virus and three different mutants showed no general correlation between mechanical properties of unfolding and the variations in frameshifting efficiency (26). However, this second study revealed that at close to 24 pN of force, the frameshifting pseudoknots exhibited an inversely proportional relationship between frameshifting efficiency and the rate constants of unfolding (26).…”

“…However, this second study revealed that at close to 24 pN of force, the frameshifting pseudoknots exhibited an inversely proportional relationship between frameshifting efficiency and the rate constants of unfolding (26).…”

“…The structural characteristics of a pseudoknot that determine frameshifting efficiency may be related to its mechanical stability and resistance to unfolding during translation (26). The possibility that the -1 PRF is dependent on mechanical tension induced by resistance introduced by a folded pseudoknot is a component of a working "torsional restraint" model (21) and further supported by recent cryoelectron microscopic imaging of ribosomes complexed with an mRNA pseudoknot (25).…”

“…These data suggest that the regulation of ribosomal frameshifting may not be simply due to thermodynamics. It has been proposed that the kinetics of unfolding may dictate frameshifting efficiencies (16), and these properties have just begun to be characterized (26,28). In addition to thermodynamic and/or kinetic control, there is evidence that the pseudoknot makes contacts with the ribosome in specific regions of the pseudoknot (29).…”

“…It is the general consensus of the field that more data from a variety of pseudoknot systems need to be studied in order to better attribute thermodynamic, kinetic, and direct interaction contributions from pseudoknot structure to overall -1 frameshifting efficiency (26,28,72). The Beet Western…”