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

Abstract

Enzymes generally are thought to derive their functional activity from conformational motions. The limited chemical variation in RNA suggests that such structural dynamics may play a particularly important role in RNA function. Minimal hammerhead ribozymes are known to cleave efficiently only in~10-fold higher than physiologic concentrations of Mg 2+ ions. Extended versions containing native loop-loop interactions, however, show greatly enhanced catalytic activity at physiologically relevant Mg 2+ concentrations, for reasons that are still ill-understood. Here, we use Mg 2+ titrations, activity assays, ensemble, and single molecule fluorescence resonance energy transfer (FRET) approaches, combined with molecular dynamics (MD) simulations, to ask what influence the spatially distant tertiary loop-loop interactions of an extended hammerhead ribozyme have on its structural dynamics. By comparing hammerhead variants with wild-type, partially disrupted, and fully disrupted loop-loop interaction sequences we find that the tertiary interactions lead to a dynamic motional sampling that increasingly populates catalytically active conformations. At the global level the wild-type tertiary interactions lead to more frequent, if transient, encounters of the loop-carrying stems, whereas at the local level they lead to an enrichment in favorable in-line attack angles at the cleavage site. These results invoke a linkage between RNA structural dynamics and function and suggest that loop-loop interactions in extended hammerhead ribozymes-and Mg 2+ ions that bind to minimal ribozymes-may generally allow more frequent access to a catalytically relevant conformation(s), rather than simply locking the ribozyme into a single active state.

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

“…The two simplest explanations for this lack of electron density are that a significant fraction of molecules degraded during crystal growth or that the crystal contains a conformational ensemble with some flexibility and/or disparity around the active site, a possibility favored by the authors (Chen et al 2010). To test this notion and provide a window into the rapid (sub-microsecond), small-scale dynamics of the ribozyme, not easily accessible by experimental techniques (Ditzler et al 2010), we performed MD simulations on the crystallized trans-acting precursor using our established protocols (Krasovska et al 2005(Krasovska et al , 2006Rhodes et al 2006;McDowell et al 2007McDowell et al , 2010Sefcikova et al 2007a,b;Ditzler et al 2010). We used the latest parmbsc0χ OL3 (Perez et al 2007;Zgarbova et al 2011) variant of the Cornell et al AMBER force field (Cornell et al 1995), which is essential for stable RNA simulations (Banas et al 2010).…”

“…Protonation of C75, in turn, leads to a C75H+(N3)…G1(O5 ′ ) hydrogen bond that aids in adopting a favorable in-line fitness. It appears that it is the required temporal coincidence of these partially competing, dynamic low-probability protonation and conformational change events that slows RNA self-cleavage to, at best, some 10s to 100s min −1 (Sefcikova et al 2007b;McDowell et al 2010). Future studies will likely reveal more such examples for the complex relationship between the folding dynamics and function of RNA.…”

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

“…smFRET has been widely used to study the ion-induced conformational changes of functional RNAs (Zhuang et al 2000;Kobitski et al 2007;Qu et al 2008;Steiner et al 2008;Xiao et al 2008;Brenner et al 2010;McDowell et al 2010;Haller et al 2011). In this study, we investigated the effect of Mg 2+ on the folding and conformational change of pRNA 3WJ using smFRET.…”

Folding of the hammerhead ribozyme: Pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

“…For clarity of the graph, the cleavage values for diAP8-diAP12 were incremented by 0.05 pH unit. McDowell et al 2010) failed to identify the twostep folding is because under physiological conditions, both global and core folding are very fast (in the millisecond range) and divided on the time scale by only a few seconds. When Mg 2+ is fully saturating, the observed rate constant of the core folding tends toward the intrinsic rate constant of the global transition.…”

“…ions that bind to minimal ribozymes may generally allow more frequent access to a catalytically relevant conformation(s), rather than simply locking the ribozyme into a single active state (McDowell et al 2010). Strikingly, both minimal and 1 natural hammerhead ribozyme catalysis requires significantly higher Mg 2+ concentrations than those required for tertiary folding (Rueda et al 2003).…”