Single-molecule enzymology of RNA: essential functional groups impact catalysis from a distance - PubMed (original) (raw)

Single-molecule enzymology of RNA: essential functional groups impact catalysis from a distance

David Rueda et al. Proc Natl Acad Sci U S A. 2004.

Abstract

The hairpin ribozyme is a minimalist paradigm for studying RNA folding and function. In this enzyme, two domains dock by induced fit to form a catalytic core that mediates a specific backbone cleavage reaction. Here, we have fully dissected its reversible reaction pathway, which comprises two structural transitions (docking/undocking) and a chemistry step (cleavage/ligation), by applying a combination of single-molecule fluorescence resonance energy transfer (FRET) assays, ensemble cleavage assays, and kinetic simulations. This has allowed us to quantify the effects that modifications of essential functional groups remote from the site of catalysis have on the individual rate constants. We find that all ribozyme variants show similar fractionations into effectively noninterchanging molecule subpopulations of distinct undocking rate constants. This leads to heterogeneous cleavage activity as commonly observed for RNA enzymes. A modification at the domain junction additionally leads to heterogeneous docking. Surprisingly, most modifications not only affect docking/undocking but also significantly impact the internal chemistry rate constants over a substantial distance from the site of catalysis. We propose that a network of coupled molecular motions connects distant parts of the RNA with its reaction site, which suggests a previously undescribed analogy between RNA and protein enzymes. Our findings also have broad implications for applications such as the action of drugs and ligands distal to the active site or the engineering of allostery into RNA.

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Figures

Fig. 1.

Dissecting the impact of single functional group modifications on hairpin ribozyme catalysis. (A) Secondary and tertiary structure of the docked WT ribozyme used in this study (9). (Left) Watson–Crick and noncanonical base pairs are indicated as solid and dashed lines, respectively. Orange, substrate; cyan arrow, cleavage site; color-coded nucleotides and junction connection were modified in this study (purple, dC12; red, dA38; green, C39S3; blue, RzAS3). (Right) 3D ribbon-and-stick representation with same color coding (

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). Yellow arrows, modified functional groups distant from the (cyan) cleavage site; pink dashed tubes, modified hydrogen bonds. (B) Schematic of the reaction pathway. Gray boxes, observed effects of the color-coded functional group modifications on individual rate constants relative to those of the WT; black box, effects on the overall cleavage rate constant.

Fig. 3.

Experimental and simulated cleavage time courses of WT and variant ribozymes. Analytical kinetic simulations (black lines) are least-squares fits to the experimental data (open circles). The derived chemistry rate constants for cleavage and ligation vary significantly with the modification. The subpopulation contributions are also included (I, light gray dashed line; II, gray dash-dotted line; III, dark gray short-dashed line; IV, black dashed line), except for the many minor subpopulations of the RzAS3-containing variants.

Fig. 2.

Exemplary single-molecule FRET time trajectories of the major subpopulations of the WT and variant hairpin ribozyme-noncleavable substrate analog complexes.

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Single-molecule enzymology of RNA: essential functional groups impact catalysis from a distance - PubMed (original) (raw)