Ribosomal frameshifting occurs when a ribosome slips a few nucleotides on an mRNA and generates a new sequence of amino acids. Programmed −1 ribosomal frameshifting (−1PRF) is used in various systems to express two or more proteins from a single mRNA at precisely regulated levels. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of −1PRF in the Escherichia coli dnaX gene. The frameshifting mRNA (FSmRNA) contained the frameshifting signals: a Shine–Dalgarno sequence, a slippery sequence, and a downstream stem loop. The dynamics of ribosomal complexes translating through the slippery sequence were characterized using smFRET between the Cy3-labeled L1 stalk of the large ribosomal subunit and a Cy5-labeled tRNALys in the ribosomal peptidyl-tRNA–binding (P) site. We observed significantly slower elongation factor G (EF-G)–catalyzed translocation through the slippery sequence of FSmRNA in comparison with an mRNA lacking the stem loop, ΔSL. Furthermore, the P-site tRNA/L1 stalk of FSmRNA-programmed pretranslocation (PRE) ribosomal complexes exhibited multiple fluctuations between the classical/open and hybrid/closed states, respectively, in the presence of EF-G before translocation, in contrast with ΔSL-programmed PRE complexes, which sampled the hybrid/closed state approximately once before undergoing translocation. Quantitative analysis showed that the stimulatory stem loop destabilizes the hybrid state and elevates the energy barriers corresponding to subsequent substeps of translocation. The shift of the FSmRNA-programmed PRE complex equilibrium toward the classical/open state and toward states that favor EF-G dissociation apparently allows the PRE complex to explore alternative translocation pathways such as −1PRF.

The sequence and secondary structure of the 5′-end of mRNAs regulate translation by controlling ribosome initiation on the mRNA. Ribosomal protein S1 is crucial for ribosome initiation on many natural mRNAs, particularly for those with structured 5′-ends, or with no or weak Shine-Dalgarno sequences. Besides a critical role in translation, S1 has been implicated in several other cellular processes, such as transcription recycling, and the rescuing of stalled ribosomes by tmRNA. The mechanisms of S1 functions are still elusive but have been widely considered to be linked to the affinity of S1 for single-stranded RNA and its corresponding destabilization of mRNA secondary structures. Here, using optical tweezers techniques, we demonstrate that S1 promotes RNA unwinding by binding to the single-stranded RNA formed transiently during the thermal breathing of the RNA base pairs and that S1 dissociation results in RNA rezipping. We measured the dependence of the RNA unwinding and rezipping rates on S1 concentration, and the force applied to the ends of the RNA. We found that each S1 binds 10 nucleotides of RNA in a multistep fashion implying that S1 can facilitate ribosome initiation on structured mRNA by first binding to the single strand next to an RNA duplex structure (“stand-by site”) before subsequent binding leads to RNA unwinding. Unwinding by multiple small substeps is much less rate limited by thermal breathing than unwinding in a single step. Thus, a multistep scheme greatly expedites S1 unwinding of an RNA structure compared to a single-step mode.

Many viruses use programmed –1 ribosomal frameshifting to express defined ratios of structural and enzymatic proteins. Pseudoknot structures in messenger RNAs stimulate frameshifting in upstream slippery sequences. The detailed molecular determinants of pseudoknot mechanical stability and frameshifting efficiency are not well understood. Here we use single-molecule unfolding studies by optical tweezers, and frameshifting assays to elucidate how mechanical stability of a pseudoknot and its frameshifting efficiency are regulated by tertiary stem-loop interactions. Mechanical unfolding of a model pseudoknot and mutants designed to dissect specific interactions reveals that mechanical stability depends strongly on triplex structures formed by stem-loop interactions. Combining single-molecule and mutational studies facilitates the identification of pseudoknot folding intermediates. Average unfolding forces of the pseudoknot and mutants ranging from 50 to 22 picoNewtons correlated with frameshifting efficiencies ranging from 53% to 0%. Formation of major-groove and minor-groove triplex structures enhances pseudoknot stem stability and torsional resistance, and may thereby stimulate frameshifting. Better understanding of the molecular determinants of frameshifting efficiency may facilitate the development of anti-virus therapeutics targeting frameshifting.

We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation-and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases—one codon—and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions.

The rugged folding-energy landscapes of RNAs often display many competing minima. How do RNAs discriminate among competing conformations in their search for the native state? By using optical tweezers, we show that the folding-energy landscape can be manipulated to control the fate of an RNA: individual RNA molecules can be induced into either native or misfolding pathways by modulating the relaxation rate of applied force and even be redirected during the folding process to switch from misfolding to native folding pathways. Controlling folding pathways at the single-molecule level provides a way to survey the manifold of folding trajectories and intermediates, a capability that previously was available only to theoretical studies.

By using optical tweezers, we have investigated the mechanical unfolding of a minimal kissing complex with only two G·C base pairs. The loop–loop interaction is exceptionally stable; it is disrupted at forces ranging from 7 to 30 pN, as compared with 14–20 pN for unfolding hairpins of 7 and 11 bp. By monitoring unfolding/folding trajectories of single molecules, we resolved the intermediates, measured their rate constants, and pinpointed the rate-limiting steps. The two hairpins unfold only after breaking the intramolecular kissing interaction, and the kissing interaction forms only after the folding of the hairpins. At forces that favor the unfolding of the hairpins, the entire RNA structure is kinetically stabilized by the kissing interaction, and extra work is required to unfold the metastable hairpins. The strong mechanical stability of even a minimal kissing complex indicates the importance of such loop–loop interactions in initiating and stabilizing RNA dimers in retroviruses.

The usual variables chemists use to affect a chemical reaction are temperature and pressure. We consider here an additional variable: force, F. By attaching a molecule to the tip of a cantilever of an atomic force microscope, or to a bead in a laser light trap, we can control the force on a single molecule. This mechanical force can drive a reaction to completion, or stabilize the reactants. Force changes the thermodynamic stability of a molecule; it can thus increase or decrease the free energy change for the reaction. Force can also speed or slow rates of reactions; it changes the free energy of activation of the reaction.

The secondary structure of a truncated P5abc subdomain (tP5abc, a 56-nucleotide RNA) of the Tetrahymena thermophila group I intron ribozyme changes when its tertiary structure forms. We have now used heteronuclear NMR spectroscopy to determine its conformation in solution. The tP5abc RNA that contains only secondary structure is extended compared with the tertiary folded form; both forms coexist in slow chemical exchange (the interconversion rate constant is slower than 1 s−1) in the presence of magnesium. Kinetic experiments have shown that tertiary folding of the P5abc subdomain is one of the earliest folding transitions in the group I intron ribozyme, and that it leads to a metastable misfolded intermediate. Previous mutagenesis studies suggest that formation of the extended P5abc structure described here destabilize a misfolded intermediate. This study shows that the P5abc RNA subdomain containing a GNRA tetraloop in P5c (in contrast to the five-nucleotide loop P5c in the tertiary folded ribozyme) can disrupt the base-paired interdomain (P14) interaction between P5c and P2.

The dimerization of viral RNA through noncovalent interactions at their 5′ ends is a key step in the life cycle of retroviruses. In Moloney murine leukemia virus, three stem-loops are important in this process. One is a self-complementary tetraloop (H1), but the other two stem-loops (H2, H3) contain highly conserved GACG tetraloops that are not self-complementary sequences. Using two-dimensional NMR, we determined the structure of the H3 stem-loop. Surprisingly, it forms a stable, homodimeric kissing complex through only two intermolecular G⋅C base pairs. Cross-strand interactions of the adenines adjacent to the intermolecular G⋅C base pairs, plus unusual strong electrostatic interactions around the base pairs, contribute to the unexpected stability. This structure shows how even stem-loops without self-complementary sequences can facilitate the intermolecular recognition between two identical RNAs, and thus initiate dimerization and encapsidation of retroviral RNAs.

An RNA kissing complex formed by the dimerization initiation site plays a critical role in the survival and infectivity of human immunodeficiency virus. Two dimerization initiation site kissing sequences, Mal and Lai, have been found in most human immunodeficiency virus 1 variants. Formation and stability of these RNA kissing complexes depend crucially on cationic conditions, particularly Mg2+. Using optical tweezers, we investigated the mechanical unfolding of single RNA molecules with either Mal-type (GUGCAC) or Lai-type (GCGCGC) kissing complexes under various ionic conditions. The force required to disrupt the kissing interaction of the two structures, the rip force, is sensitive to concentrations of KCl and MgCl2; addition of 3 mM MgCl2 to 100 mM KCl changes the rip force of Mal from 21 ± 4 to 46 ± 3 pN. From the rip force distribution, the kinetics of breaking the kissing interaction is calculated as a function of force and cation concentration. The two kissing complexes have distinct unfolding transition states, as shown by different values of ΔX‡, which is the distance from the folded structure to the unfolding transition state. The ΔX‡ of Mal is ∼ 0.6 nm smaller than that of Lai, suggesting that fewer kissing base pairs are broken at the transition state of the former, consistent with observations that the Lai-type kissing complex is more stable and requires significantly more force to unfold than the Mal type. More importantly, neither K+ nor Mg2+ significantly changes the position of the transition state along the reaction coordinate. However, increasing concentrations of cations increase the kinetic barrier. We derived a cation-specific parameter, m, to describe how the height of the kinetic barrier depends on the concentration of cations. Our results suggest that Mg2+ greatly slows down the unfolding of the kissing complex but has moderate effects on the formation kinetics of the structure.

By exerting mechanical force, it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles, and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article. The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that the longest handles and softest traps that allow detection of the folding/unfolding signal (handles ∼5–10 Kbp and traps ∼0.03 pN/nm) represent the best conditions to obtain the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules.

Experimental variables of optical tweezers instrumentation that affect RNA folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was trapped by dual-beam lasers and the other was held by a micropipette. Several experimental variables were changed while measuring the unfolding/refolding kinetics, including handle lengths, trap stiffness, and modes of force applied to the molecule. In constant-force mode where the tension applied to the RNA was maintained through feedback control, the measured rate coefficients varied within 40% when the handle lengths were changed by 10-fold (1.1–10.2 Kbp); they increased by two- to threefold when the trap stiffness was lowered to one-third (from 0.1 to 0.035 pN/nm). In the passive mode, without feedback control and where the force applied to the RNA varied in response to the end-to-end distance change of the tether, the RNA hopped between a high-force folded-state and a low-force unfolded-state. In this mode, the rates increased up to twofold with longer handles or softer traps. Overall, the measured rates remained with the same order-of-magnitude over the wide range of conditions studied. In the companion article on pages 3010–3021, we analyze how the measured kinetics parameters differ from the intrinsic molecular rates of the RNA, and thus how to obtain the molecular rates.