Ensemble simulations: folding, unfolding and misfolding of a high-efficiency frameshifting RNA pseudoknot

Abstract

Massive all-atom molecular dynamics simulations were conducted across a distributed computing network to study the folding, unfolding, misfolding and conformational plasticity of the high-efficiency frameshifting double mutant of the 26 nt potato leaf roll virus RNA pseudoknot. Our robust sampling, which included over 40 starting structures spanning the spectrum from the extended unfolded state to the native fold, yielded nearly 120 μs of cumulative sampling time. Conformational microstate transitions on the 1.0 ns to 10.0 μs timescales were observed, with post-equilibration sampling providing detailed representations of the conformational free energy landscape and the complex folding mechanism inherent to the pseudoknot motif. Herein, we identify and characterize two alternative native structures, three intermediate states, and numerous misfolded states, the latter of which have not previously been characterized via atomistic simulation techniques. While in line with previous thermodynamics-based models of a general RNA folding mechanism, our observations indicate that stem-strand-sequence-separation may serve as an alternative predictor of the order of stem formation during pseudoknot folding. Our results contradict a model of frameshifting based on structural rigidity and resistance to mechanical unfolding, and instead strongly support more recent studies in which conformational plasticity is identified as a determining factor in frameshifting efficiency.

Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot

“…Computational studies have predicted that PKs fold through diverse mechanisms, depending on their sequence, by parallel pathways, cooperatively in an all-or-none manner, or by a combination of both mechanisms, exploring several intermediate states, some of which contain nonnative base pairing or alternate structures ( 18 , 41 – 47 ). The principle that assembly routes of PKs depend on the stabilities of individual secondary structural elements ( 18 ) explains the diversity of the folding mechanisms of PK.…”

Plasmon Rulers as a Probe for Real-Time Microsecond Conformational Dynamics of Single Molecules

“…However, many biomolecules exhibit short-lived intermediates in their folding trajectory with microsecond lifetimes; prime examples are metastable intermediates of small folding proteins, 49 partially open intermediates associated with membrane transporters, 50 and pseudoknots in oligonucleotides. 51 Such metastable states have typical lifetimes of some tens of microseconds but cannot be detected directly. In Figure 5a, we show a simulated timetrace of an oligonucleotide that exchanges between three conformations on microsecond time scales.…”

Programmed −1 ribosomal frameshifting from the perspective of the conformational dynamics of mRNA and ribosomes

“…Remarkably, the WNV stimulatory structure, which causes an extremely high frameshifting efficiency (up to 70%) [27] , shows extensive conformational plasticity [32] . The alternative folding propensity of stimulatory structures has also been identified in other viruses, such as the turnip crinkle virus [28] , the potato leaf roll virus [30] , and the recently identified SARS-CoV-2 [33] , [90] , through various methods. These results suggest that conformational plasticity is a common feature for most, if not all, frameshift-stimulating RNA structures.…”

“…An increasing body of evidence has shown that in addition to structural stability, the conformational dynamics of an mRNA structure play a key role in the stimulatory effects on ribosomal frameshifting [24] , [25] , [26] , [27] , [28] , [29] , [30] , [31] , [32] , [33] . In addition, ribosomes and tRNA also undergo profound conformational changes during translocation [34] , [35] , though how their structures are involved in −1 PRF remain unclear.…”