Rescue of an Abasic Hairpin Ribozyme by Cationic Nucleobases

Lebruska, Lori L.; Kuzmine, I; Fedor, Martha J.

doi:10.1016/s1074-5521(02)00130-8

Cited by 65 publications

(108 citation statements)

References 49 publications

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“…Water was not observed in the active site of previous 4WJ hairpin ribozyme structures, although solvent has been invoked to account for both structural and biochemical observations regarding the mechanism of action (10,(15)(16)(17). In earlier structural studies, it was noted that the N1 imino position of G8 appeared to function as an obligate proton donor to the 2'-oxygen of the nucleophile, and not as a proton acceptor as corroborated here for AP8 and DAP8.…”

Section: Implications For Water In the Active Sitementioning

confidence: 57%

“…The first entails acid/base catalysis involving G8 and/or A38 (14,23,52). The second calls for electrostatic stabilization involving localized nucleobase charge dissipation (15)(16)(17)23,53). Although X-ray crystallography cannot differentiate between these proposals, it can provide additional stereochemical information relevant to understanding the principles by which the enzyme promotes catalytically productive or non-productive states.…”

Section: Discussionmentioning

confidence: 99%

“…Further evidence suggesting the importance of G8 was derived from studies of a 4WJ hairpin ribozyme in which G8 was made abasic. This modification resulted in a 350-fold loss in catalytic activity, whereas abasic substitutions at neighboring A9 and A10 positions resulted in <10-fold losses, respectively (15). Subsequent studies suggested that nucleobase rescue of abasic G8 ribozymes was consistent with electrostatic stabilization of the transition state (16).…”

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confidence: 99%

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Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

et al. 2005

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The hairpin ribozyme requires functional group contributions from G8 to assist in phosphodiester bond cleavage. Previously, replacement of G8 by a series of nucleobase variants showed little effect on interdomain docking, but a 3-to 250-fold effect on catalysis. To identify G8 features that contribute to catalysis within the hairpin ribozyme active site, structures for five base variants were solved by X-ray crystallography in a resolution range between 2.3 to 2.7 Å. For comparison, a native all-RNA "G8" hairpin ribozyme structure was refined to 2.05 Å resolution. The native structure revealed a scissile bond angle (τ) of 158°, which is close to the requisite 180° 'in-line' geometry. Mutations G8(inosine), G8(diaminopurine), G8(aminopurine), G8(adenosine) and G8(uridine) folded properly, but exhibited non-ideal scissile bond geometries (τ ranging from 118° to 93°) that paralleled their diminished solution activities. A superposition ensemble of all structures, including a previously described hairpin ribozyme-vanadate complex, indicated the scissile bond can adopt a variety of conformations resulting from perturbation of the chemical environment, and provided a rationale for how the exocyclic amine of nucleobase 8 promotes productive, in-line geometry. Changes at position 8 also caused variations in the A−1 sugar pucker. In this regard, variants A8 and U8 appeared to represent non-productive ground-states in which their 2'-OH groups mimicked the pro-R, non-bridging oxygen of the vanadate transition-state complex. Finally, the results indicated that ordered water molecules bind near the 2'-hydroxyl of A−1, lending support to the hypothesis that solvent may play an important role in the reaction. † This work was supported by NIH Grant GM63162 to J.E.W. ‡ Protein Data Bank Codes for the reported structures: 1ZFR (G8), 1ZFT (G8I), 1ZFV (G8A), 1ZFX (G8U), 2BCY (G8AP), 2BB1 (G8DAP), 2BCZ (G8I/dA−1). § Present address: Rosalind Franklin School of Science and Medicine, Dept. Biochem. and Mol. Biol., 3333 Green Bay Road Rd., N. Chicago, IL 60064, USA * To whom correspondence should be addressed: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Box 712, Rochester, New York 14642 USA. Phone: 585 273-4516; Fax: 585 275-6007; Email: Joseph_Wedekind@URMC.Rochester.edu ∥ These authors contributed equally to this work.Supporting Information Available A stereo diagram of representative electron density maps is provided for the native and position 8 variants of this study (Figure 1). A diagram comparing the G8I/2'-OMe A−1 and G8I/2'-deoxy A−1 variants is also available (Figure 2). The method for HPLC composition analysis of AP8 and DAP8 crystals is reported, as well as elution profiles for the separated hairpin ribozyme strands (Figure 3). This information is provided free of charge at http://pubs.acs.org NIH Public Access The hairpin ribozyme is a small ribozyme whose family members catalyze a reversible, sitespecific phosphodiester bond cleavage reacti...

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Section: Implications For Water In the Active Sitementioning

confidence: 57%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

et al. 2005

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“…The nucleobase could stabilize the reaction transition state by specific hydrogen bonding, or a protonated adenine might stabilize the dianionic phosphorane electrostatically. Both effects have been proposed for the hairpin ribozyme (3,21). Were this to be the case in the VS ribozyme, then substitution of the nucleobase at position 756 could lead to lower cleavage rates, as observed.…”

Section: Discussionmentioning

confidence: 75%

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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“…However, recent crystallographic and biochemical experiments have suggested that only two nucleobases in the ribozyme, G8 and A38, are directly involved in reaction chemistry ( Fig. 1 A) (9)(10)(11)(12). An open question is therefore how all of the other functional groups exert their influence on activity.…”

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confidence: 99%

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

Rueda

Bokinsky

Rhodes

et al. 2004

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

138

200

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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 (docking͞undocking) and a chemistry step (cleavage͞ligation), 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 docking͞undocking 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. R NA enzymes (ribozymes) have been recognized as ideal model systems for studying the relationship of structure and function in RNA, because their catalytic activity directly reports the extent of native structure formation (1-3). This provides the basis for powerful modification-interference experiments in which the activity of site-specifically modified ribozymes is compared to the unmodified WT to map functionally important residues of the catalytic core. Such chemical modifications, however, may impact ribozyme function through reaction chemistry, structure formation, or both. Distinguishing these mechanisms has long been an experimental challenge. A typical example is the hairpin ribozyme, derived from the self-cleaving 359-nt negative strand of the tobacco ringspot virus satellite RNA, a member of a family of plant pathogens (4-6). A wealth of modification-interference experiments has helped to define the residues important for function of the minimal two-way junction form of this catalytic RNA, designed as the sequence with highest enzymatic activity in external substrate cleavage (4,5,7,8). Many functional groups of the 24 non-Watson-Crick base-paired nucleotides in the two internal loops of domains A and B were shown to be essential for catalytic activity (Fig. 1A). However, recent crystallographic and biochemical experiments have suggested that only two nucleobases in the ribozyme, G8 and A38, are directly involved in reaction chemistry (Fig. 1 A) (9-12). An...

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Rescue of an Abasic Hairpin Ribozyme by Cationic Nucleobases

Cited by 65 publications

References 49 publications

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

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

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

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Rescue of an Abasic Hairpin Ribozyme by Cationic Nucleobases

Cited by 65 publications

References 49 publications

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer,

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer,

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

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

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Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,

Water in the Active Site of an All-RNA Hairpin Ribozyme and Effects of Gua8 Base Variants on the Geometry of Phosphoryl Transfer^,