Folding of the Hairpin Ribozyme in Its Natural Conformation Achieves Close Physical Proximity of the Loops

Murchie, Alastair I. H.; Thomson, John F.; Walter, Frank; Lilley, David M.J.

doi:10.1016/s1097-2765(00)80086-6

Cited by 154 publications

(164 citation statements)

References 47 publications

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“…It is likely that an association of the substrate loop with the A730 loop of the ribozyme would generate the structure required for catalytic activity. A similar situation exists in the hairpin ribozyme, where the active geometry results from a close interaction between the A and B loops (23). The local structure (3,24) is significantly altered from those of the loops in isolation (25,26).…”

Section: Discussionmentioning

confidence: 73%

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Wilson

Lü

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

120

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Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK a ¼ 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5′ oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolatemodified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2′-O nucleophile by G638 and protonation of the 5′-O leaving group by A756. 5′-phosphorothiolate | RNA catalysis | nucleolytic ribozymes | catalytic mechanism R ibozyme-mediated catalysis is important for both RNA splicing and translation (1), yet its chemical origins are incompletely understood. The nucleolytic ribozymes bring about the site-specific cleavage or ligation of RNA, with an acceleration of a millionfold or greater. The intensively studied protein RNase A catalyses an identical cleavage reaction, and much evidence supports the hypothesis that each of these phosphoryl transfer reactions is subject to general acid-base catalysis. This mechanism requires a general base to deprotonate the attacking nucleophile, and a general acid to protonate the oxyanion leaving group (Fig. 1).The most common chemical entities implicated in RNA catalysis by the nucleolytic ribozymes are the nucleobases (2). Guanine appears to play a catalytic role in the hairpin, hammerhead, GlmS, and Varkud satellite (VS) ribozymes, adenine in the hairpin, and VS and cytosine in the hepatitis delta virus (HDV) ribozyme. Crystal structures of the hairpin ribozyme (3) reveal the presence of guanine (G8) and adenine (A38) bases juxtaposed with the 2′-O and 5′-O, respectively, of the scissile phosphate, where they seem poised to act in general acid-base catalysis. This is consistent with the pH dependence of the reaction (4) and its variation with functional group modifications (5-8).In its simplest active form, the VS ribozyme comprises five helices (II through VI) organized by two three-way junctions, which acts in trans upon a substrate stem loop (helix I) with an internal loop that contains the scissile phosphate (Fig. 1). The loop also contains the critical G638 (9). A756 (10-13) is contained within an internal loop in helix VI. While no crystal structure of the VS ribozyme has yet been solved, a small-angle X-ray scattering-derived model plac...

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Section: Discussionmentioning

confidence: 73%

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Wilson

Lü

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

120

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“…Although the ribozyme is active in the absence of the junction, 16 its presence accelerates the ion-induced folding of the ribozyme by 500-fold, 5 and reduces the required ionic concentration by three orders of magnitude and thus allows folding to proceed under physiological conditions. 1,2,4 We have therefore sought to understand the conformational properties of this junction in the absence of the loops, i.e. as a simple 4H junction.…”

Section: Introductionmentioning

confidence: 99%

Conformational Flexibility of Four-way Junctions in RNA

Hohng

Wilson

Tan

et al. 2004

Journal of Molecular Biology

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Helical junctions are common architectural features in RNA. They are particularly important in autonomously folding molecules, as exemplified by the hairpin ribozyme. We have used single-molecule fluorescence spectroscopy to study the dynamic properties of the perfect (4H) four-way helical junction derived from the hairpin ribozyme. In the presence of Mg 2þ , the junction samples parallel and antiparallel conformations and both stacking conformers, with a bias towards one antiparallel stacking conformer. There is continual interconversion between the forms, such that there are several transitions per second under physiological conditions. Our data suggest that interconversion proceeds via an open intermediate with reduced cation binding in which coaxial stacking between helices is disrupted. The rate of interconversion becomes slower at higher Mg 2þ concentrations, yet the activation barrier decreases under these conditions, indicating that entropic effects are important. Transitions also occur in the presence of Na þ only; however, the coaxial stacking appears incomplete under these conditions. The polymorphic and dynamic character of the four-way RNA junction provides a source of structural diversity, from which particular conformations required for biological function might be stabilised by additional RNA interactions or protein binding.

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“…7]. The reaction mechanism of the hairpin ribozyme has been explored by ensemble kinetic and structural studies (6)(7)(8)(9)(10)(11)(12)(13)(14)(15) and singlemolecule experiments (16)(17)(18)(19)(20)(21). The enzyme catalyzes the cleavage reaction in several steps (Fig.…”

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confidence: 99%

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

Liu¹,

Bokinsky²,

Walter

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

123

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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...

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Folding of the Hairpin Ribozyme in Its Natural Conformation Achieves Close Physical Proximity of the Loops

Cited by 154 publications

References 47 publications

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Conformational Flexibility of Four-way Junctions in RNA

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

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