Single VS Ribozyme Molecules Reveal Dynamic and Hierarchical Folding Toward Catalysis

Abstract

Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two threeway junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.

Conformational change in the Bacillus subtilis RNase P holoenzyme–pre-tRNA complex enhances substrate affinity and limits cleavage rate

“…All of these processes could occur in the observed conformational change that enhances the affinity of B. subtilis RNase P for pretRNA. Previous observations of other ribozymes, including the hairpin ribozyme (Walter et al 1998;Wilson et al 2005), the SV ribozyme (Pereira et al 2008), and Group I intron (for a recent review, see Hougland et al 2006) demonstrate that substrates first bind to the enzyme in a low-affinity initial complex in an ''open'' conformation, which then isomerizes to a high-affinity conformational state as a ''closed'' or ''docked'' state. The fluorescence binding data are the first explicit demonstration that RNase P similarly uses a two-step substrate binding mechanism that enhances both substrate binding and catalysis.…”

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations

“…To analyze our data, we first plotted aggregate FRET histograms for each set of conditions and fit them with Gaussians to identify FRET states ( Fig. 4A-C For a relative comparison, we mapped the distances derived from the tr-FRET analysis onto the FRET ratios of our sm-FRET derived histograms, using similar strategies and assumptions as described previously (Pereira et al 2008). This comparison shows the general consistency between the two types of data.…”

“…Single-molecule experiments were performed as previously described (Rueda et al 2004;Pereira et al 2008;de Silva and Walter 2009). The biotinylated and Cy3-Cy5-labeled ribozyme and noncleavable substrate strands ( Fig.…”

“…The annealed complex was diluted to <20 pM and bound to a streptavidin-coated quartz slide surface via the biotinstreptavidin interaction. The donor (I D ) and acceptor (I A ) fluorescence signals of optically resolved single molecules (characterized by single-step photobleaching) were detected on a total internal reflection fluorescence microscope as described (Rueda et al 2004;Pereira et al 2008). The FRET ratio [defined as I A /(I A + I D )] was followed in real time for each individual molecule.…”

“…The apparent FRET efficiency was calculated using a scaling factor c = 0.68 as in Sabanayagam et al (2005), then the FRET ratio was calculated from this apparent FRET efficiency (Lakowicz 2006), in an analogous manner to previously described calculations (Pereira et al 2008). Since our observed sm-FRET ratio is z0.2 in the absence of acceptor fluorophore, this is the lower limit of our calculations.…”