Dissecting the multistep reaction pathway of an RNA enzyme by single-molecule kinetic “fingerprinting”

Abstract

Single-molecule FRET is a powerful tool for probing the kinetic mechanism of a complex enzymatic reaction. However, not every reaction intermediate can be identified via a distinct FRET value, making it difficult to fully dissect a multistep reaction pathway. Here, we demonstrate a method using sequential kinetic experiments to differentiate each reaction intermediate by a distinct time sequence of FRET signal (a kinetic ''fingerprint''). Our model system, the two-way junction hairpin ribozyme, catalyzes a multistep reversible RNA cleavage reaction, which comprises two structural transition steps (docking/undocking) and one chemical reaction step (cleavage/ligation). Whereas the docked and undocked forms of the enzyme display distinct FRET values, the cleaved and ligated forms do not. To overcome this difficulty, we used Mg 2؉ pulsechase experiments to differentiate each reaction intermediate by a distinct kinetic fingerprint at the single-molecule level. This method allowed us to unambiguously determine the rate constant of each reaction step and fully characterize the reaction pathway by using the chemically competent enzyme-substrate complex. We found that the ligated form of the enzyme highly favors the docked state, whereas undocking becomes accelerated upon cleavage by two orders of magnitude, a result different from that obtained with chemically blocked substrate and product analogs. The overall cleavage reaction is rate-limited by the docking/undocking kinetics and the internal cleavage/ligation equilibrium, contrasting the rate-limiting mechanism of the four-way junction ribozyme. These results underscore the kinetic interdependence of reversible steps on an enzymatic reaction pathway and demonstrate a potentially general route to dissect them. fluorescence resonance energy transfer ͉ hairpin ribozyme ͉ reaction kinetics ͉ ribozyme E nzymatic reactions often involve multiple kinetic steps such as substrate binding, folding of the enzyme-substrate complex, catalytic chemistry, and product dissociation. Because of the difficulty associated with the isolation of each intermediate species of the reaction, it is a challenging task to determine the entire set of microscopic rate constants that constitute such a multistep reaction scheme. Monitoring the reaction of a single molecule can potentially alleviate this problem. As an example, FRET has been exploited to probe conformational changes of single molecules in real time, making it well suited for monitoring structural intermediates (1-3). However, chemical reaction intermediates typically differ by only one or a few covalent bonds and often cannot be distinguished directly by the distancesensitive probing based on FRET. Here, we demonstrate a kinetic ''fingerprint'' strategy to overcome this difficulty by using an RNA enzyme as a model system.The hairpin ribozyme catalyzes a reversible site-specific RNA cleavage reaction (4, 5). The enzyme consists of two helix-loophelix domains, with the cleavage site located within the substrate strand that makes up half...

Kinetic Analysis of Sequential Multistep Reactions

“…over the entire observation time window). The uncertainty in p(t) arises from the Poisson counting noise and thus the variance of n τ (t) is equal to its mean: (10) From this, it is clear that we cannot naively estimate m~d ln p(t)/d lnt, t → 0 + , due to the numerical uncertainty (Note: n τ (t) ∝ p(t) ∝ t m−1 → 0, as t → 0 + for multi-step reactions where m > 1).…”

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

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“…However, the entropic cost of ligation is rather small and can be compensated by the favorable enthalpic contribution (Hegg andFedor 1995, Nahas et al 2004). In addition, ligation is about two times faster than cleavage (Liu et al 2007). Thus, the internal equilibrium of the hairpin ribozyme is shifted toward ligation.…”