Mimicking Ribosomal Unfolding of RNA Pseudoknot in a Protein Channel

Abstract

Pseudoknots are a fundamental RNA tertiary structure with important roles in regulation of mRNA translation. Molecular force spectroscopic approaches such as optical tweezers can track the pseudoknot’s unfolding intermediate states by pulling the RNA chain from both ends, but the kinetic unfolding pathway induced by this method may be different from that in vivo, which occurs during translation and proceeds from the 5′ to 3′ end. Here we developed a ribosome-mimicking, nanopore pulling assay for dissecting the vectorial unfolding mechanism of pseudoknots. The pseudoknot unfolding pathway in the nanopore, either from the 5′ to 3′ end or in the reverse direction, can be controlled by a DNA leader that is attached to the pseudoknot at the 5′ or 3′ ends. The different nanopore conductance between DNA and RNA translocation serves as a marker for the position and structure of the unfolding RNA in the pore. With this design, we provided evidence that the pseudoknot unfolding is a two-step, multistate, metal ion-regulated process depending on the pulling direction. Most notably, unfolding in both directions is rate-limited by the unzipping of the first helix domain (first step), which is Helix-1 in the 5′ → 3′ direction and Helix-2 in the 3′ → 5′ direction, suggesting that the initial unfolding step in either pulling direction needs to overcome an energy barrier contributed by the noncanonical triplex base-pairs and coaxial stacking interactions for the tertiary structure stabilization. These findings provide new insights into RNA vectorial unfolding mechanisms, which play an important role in biological functions including frameshifting.

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

“…The absence of a correlation is consistent with previous observations37 and is likely due to the fact that the formation of stem 2 and minor-groove triples (formed between stem 1 and loop 2) in a pseudoknot may exert a coupling effect in stabilizing stem 1 as has been observed in the recent nanopore pseudoknot unzipping experiments45. The thermodynamic stabilization effect due to the coaxial stacking between stem 1 and stem 2 and stem 1-loop 2 bases triple formation are not observed in the thermal denaturation, because under the global temperature perturbation, the base triples and stem 2 melt before stem 1 (Fig.…”

“…Such a coupling effect of stem-loop interactions in stabilizing stem 1 has been observed in the recent nanopore pseudoknot unzipping experiments45.…”

Molecular mechanisms underlying the extreme mechanical anisotropy of the flaviviral exoribonuclease-resistant RNAs (xrRNAs)

“…To facilitate directional translocation of ZIKV xrRNA1 through the -hemolysin (-HL) nanopore, a 36-nt poly(rA) leader sequence is co-transcribed with the xrRNA1 at the 5' or 3' end (hereafter as 5'A36-xrRNA1 and 3'A36-xrRNA1, respectively) (Figure 2a and b). Similar designs have been used to facilitate directional translocation of the human telomere i-motif DNA and T2 pseudoknot RNA through -HL nanopore 19,26 . DSC experiments on these xrRNA1s show that the leader sequence has minor effects on the thermal stability of xrRNA1s ( Figure S4, Table S2).…”

“…Initial nanopore sensing studies were conducted for the ZIKV xrRNA1 sequences in 1M KCl supplemented with 5 mM MgCl2 (cis side) at +100 mV. Under such condition, the force applied to leader sequence is around 8 pN 19 , close to that exerted by molecular machines 27,28 . Figure 2c shows a representative current blockade signature for the 5'A36-xrRNA1 through the nanopore, which only very long current blockade is observed.…”

“…By pulling the RNAs from both ends, these approaches can identify the intermediate states along the folding pathway, but they also have obvious limitations in which the unfolding process is not unidirectional, differ from that in vivo. Fortunately, recent developments in single-molecule nanopore sensing technology have allowed the unfolding and translocation of RNAs through biological pores in a defined direction to be observed with fine details 18,19,20 . By taking advantage of the ability to electrically detect charged biomolecules through a nanometer-wide channel, several types of nanopores including -hemolysin (-HL) nanopore have been developed 21,22 .…”