RNA reactions one molecule at a time

Abstract

Much of the dynamics information is lost in bulk measurements because of the population averaging. Single-molecule methods measure one molecule at a time; they provide knowledge not obtainable by other means. In this article, we review the application of the two most widely used single-molecule methods--fluorescence resonance energy transfer (FRET) and force versus extension measurements--to several RNA reactions. First, we discuss folding/unfolding studies on a hairpin ribozyme that revealed multiple conformations of the RNA with distinct kinetics, and on a series of RNA pseudoknots, whose mechanical stabilities were found to show a strong correlation with their frameshifting efficiency during translation. We also discuss several RNA-related molecular motors. Single-molecule experiments revealed detailed mechanisms for the interaction of HIV reverse transcriptase and nucleic acid helicases (NS3 and RIG-1) with their substrates. Optical tweezers studies showed that translation of a single messenger RNA by a ribosome occurs by successive translocation-and-pause cycles. Single-molecule FRET experiments yielded important information on ribosome conformational changes and tRNA dynamics during translation. Overall, single-molecule experiments have been very valuable for understanding RNA reactions.

Figure 1.

Optical tweezers assay for studying NS3 helicase unwinding or ribosome translation. (A) The RNA has a short single-stranded region for loading NS3 helicase or ribosome, followed by a hairpin region to be unwound or translated. Note that NS3 translocates 3′ to 5′, whereas the ribosome translates mRNA 5′ to 3′. The two ends of the RNA are attached to long handles for separating the RNA/enzyme construct from the beads. The ends are then held between two micron-size polystyrene beads coated with antidigoxigenin antibody and streptavidin. Drawings are schematic and not to scale. (B, C) Representative traces of extension versus time for NS3 unwinding (B; force is maintained at 15 pN) and ribosome translation (C; force is maintained at 20 pN). Adapted from Wen et al. 2008 and Dumont et al. 2006.

Figure 2.

Pseudoknots and –1 ribosomal frameshifting. (A) Pseudoknots used for bulk frameshift assays and single-molecule studies. The pseudoknots contain stem 1 (red), loop 1 (yellow), stem 2 (blue), and loop 2 (green). All the mutants were made based on ΔU177. In mutant CCCGU, all base triples are disrupted (two in stem 1-loop 2 and three in stem 2-loop 1). In TeloWT, the single nucleotide bulge U177 in stem 2 is added. The directions of applied mechanical force by optical tweezers are shown with black arrows. (B) Correlation observed between bulk –1 frameshifting efficiency and average unfolding force. Error bars are standard deviations from bulk and single-molecule experiments. Adapted from Chen et al. 2009.

Figure 3.

Single-molecule FRET assay for probing the binding mechanism of HIV-1 reverse transcriptase (RT) on nucleic acid substrates. (A) The structure of HIV-1 RT bound to a substrate. RT is labeled with Cy3 at one of the two labeling sites (green stars). (B) The substrate consists of a 19–21-nt primer strand (DNA or RNA) annealed to a 50-nt DNA template strand to mimic the different binding substrates that RT encounters in vivo. The template is labeled with a Cy5 dye (red star) at either the 5′ or 3′ end. (C) One end of the substrate is immobilized on the coverslip surface for single-molecule detection. The RT is free to diffuse in solution. Under 532 nm excitation wavelength in a total internal reflection fluorescence (TIRF) microscospe, no fluorescence is detected without RT binding; when an RT binds to the substrate, the donor dye is excited by the evanescent laser illumination and the FRET signal of the dye pair can be observed until the RT dissociates from the substrate or the dyes photobleach. The stars and spheres indicate dyes that do and do not emit fluorescence, respectively. (D) An example of smFRET analysis. Top: fluorescence time traces from Cy3 (green) and Cy5 (red) under excitation at 532 nm, and that from Cy5 (pink) under excitation at 635 nm (to confirm that Cy5 has not photobleached). Middle: FRET value calculated over the duration of the binding events (yellow shaded regions). Bottom: FRET distribution histogram created for the binding events. The observed FRET value and its change over time report the RT binding orientation, and the dynamics of RT orientation change, respectively. The same experiment was repeated using different FRET labeling sites on the RT and/or substrate to strengthen the conclusions. (Reprinted, with permission, from Abbondanzieri et al. 2008 [© MacMillan].)