Single-Molecule Mechanical Folding and Unfolding of RNA Hairpins: Effects of Single A-U to A·C Pair Substitutions and Single Proton Binding and Implications for mRNA Structure-Induced −1 Ribosomal Frameshifting

Abstract

A wobble A·C pair can be protonated at near physiological pH to form a more stable wobble A·C pair. Here, we constructed an RNA hairpin (rHP) and three mutants with one A-U base pair substituted with an A·C mismatch on the top (near the loop, U22C), middle (U25C), and bottom (U29C) positions of the stem, respectively. Our results on single-molecule mechanical (un)folding using optical tweezers reveal the destabilization effect of A-U to A·C pair substitution and protonation-dependent enhancement of mechanical stability facilitated through an increased folding rate, or decreased unfolding rate, or both. Our data show that protonation may occur rapidly upon the formation of an apparent mechanical folding transition state. Furthermore, we measured the bulk -1 ribosomal frameshifting efficiencies of the hairpins by a cell-free translation assay. For the mRNA hairpins studied, -1 frameshifting efficiency correlates with mechanical unfolding force at equilibrium and folding rate at around 15 pN. U29C has a frameshifting efficiency similar to that of rHP (∼2%). Accordingly, the bottom 2-4 base pairs of U29C may not form under a stretching force at pH 7.3, which is consistent with the fact that the bottom base pairs of the hairpins may be disrupted by ribosome at the slippery site. U22C and U25C have a similar frameshifting efficiency (∼1%), indicating that both unfolding and folding rates of an mRNA hairpin in a crowded environment may affect frameshifting. Our data indicate that mechanical (un)folding of RNA hairpins may mimic how mRNAs unfold and fold in the presence of translating ribosomes.

Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch

“…To better define the conformational landscape of such RNAs, observing the folding and unfolding trajectories of individual molecules under tension can provide information beyond the resolution of conventional ensemble techniques, which are necessarily limited by molecular averaging (Visscher, 2016). In recent years, the application of single molecule force spectroscopy as a tool for probing structural transitions has yielded unprecedented insights into various nucleic acid structures (Chandra et al, 2017; Greenleaf et al, 2008; Mandal et al, 2019; Yang et al, 2018; Zhong et al, 2016), dynamic cellular processes (Desai et al, 2019; Wen et al, 2008) as well as mechanisms of PRF (Chen et al, 2017; Green et al, 2008; Halma et al, 2019; Yan et al, 2015).…”

Base-pair Opening Dynamics of Nucleic Acids in Relation to Their Biological Function

“…These experiments have been used to probe base-pair opening in various DNA duplexes [[3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]], DNAs containing modified base such as 5-flurourasil, N6-methyl adenine, or modified guanine [[19], [20], [21]], UV-induced photoadduct-containing DNA [22], IHF-complexed DNA [23], interstrand cross-linked DNA [24], i-motif structure formed by the complementary C-rich DNA [25,26], various RNAs [15,[27], [28], [29], [30], [31], [32], [33], [34], [35], [36]], peptide nucleic acids (PNAs) [37,38], and threose nucleic acid (TNA) [39]. NMR exchange and single molecule FRET experiments could be used to study the protonation/deprotonation of adenine bases and formation of A + ·C wobble pair [[40], [41], [42], [43], [44]]. In addition, hydrogen exchange data can also be used to probe how intermolecular interactions stabilize nucleic acid duplexes.…”

Programmed −1 Ribosomal Frameshifting in coronaviruses: A therapeutic target

“…It has been known for some time that thermodynamic stability corresponds with −1 PRF efficiency, but only to a limited extent: pseudoknots that are too stable inhibit −1 PRF, presumably because they cannot be resolved by translating ribosomes ( Marczinke et al, 1998 ). Numerous studies suggest that dynamic mRNA structural remodeling is required for optimal −1 PRF efficiency ( Chen et al, 2013 ; Halma et al, 2019 , 2020 ; Kendra et al, 2017 ; Kim et al, 2014 ; Moomau et al, 2016 ; Ritchie et al, 2012 , 2014 , 2017 ; Yang et al, 2018 ). Coordination of base triples in both major and minor grooves provides mechanical resistance to pseudoknot unwinding and stretches of adenosines confined along the minor groove of a helix also provide resistance.…”