Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer

Walter, Nils G.; Hampel, Ken J.; Brown, Kirk M.; Burke, John M.

doi:10.1093/emboj/17.8.2378

Cited by 162 publications

(218 citation statements)

References 56 publications

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“…Interestingly, the same or similar ribozymes with a noncleavable substrate (carrying a chemistryblocking 2Ј-OMe A-1 instead of 2Ј-OH A-1 at the cleavage site) has a comparable docking rate constant (0.018 s Ϫ1 ), but undocks 20-fold faster (0.01 s Ϫ1 ) (9,16,17,19). These results indicate that the structural dynamics derived from a nonreactive substrate analog do not faithfully represent those of the unmodified substrate.…”

Section: Discussionmentioning

confidence: 98%

“…3ЈP with the fluorescence quencher dabcyl (3ЈP-D) was purchased from Trilink Biotechnologies (San Diego, CA). RNA was deprotected according to the manufacturers' protocols and then purified by denaturing PAGE and C8 RP-HPLC as described (9). To assemble the ribozyme, we annealed RzA and RzB strands by heating to 90°C for 1 min and then slowly cooling to room temperature over 45 min in annealing buffer (50 mM Tris⅐HCl, pH 7.5/50 mM NaCl/1 mM EDTA).…”

Section: Methodsmentioning

confidence: 99%

“…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%

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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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Section: Discussionmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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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“…The mutations in the loop A ribozyme strand ( Figure 1A, green) were introduced at position 8 and included: G8I, G8DAP, G8AP, G8A and G8U. Mutations to the substrate strand ( Figure 1A, red) were: dA−1 and 2'-OMeA−1, which were shown to inactivate the ribozyme from cleavage without disrupting the tertiary fold (26)(27)(28). Under high pH conditions, the G8DAP and G8AP substitutions cleaved slowly (14) and were used in this study at pH > 8.5 in combination with an A−1 ribonucleotide.…”

Section: Experimental Procedures the Minimal All-rna Hairpin Ribozymementioning

confidence: 99%

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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“…Under these conditions, chemistry is ratelimiting up to approximately pH 8.5, so the rate of any conformation changes must significantly exceed 500 min -1 , the rate of the chemical step at pH 8.5. In comparison, tRNA folds 10-fold faster than this, the catalytic domain of RNase P folds at the same rate, the fast steps of group I intron folding proceed 8-fold slower, and large conformational changes of several ribozymes occur on the order of minutes (41,(44)(45)(46)(47)(48)(49).…”

Section: Role Of Metal Ions In Structure Andmentioning

confidence: 99%

Metal Ion Requirements for Structure and Catalysis of an RNA Ligase Ribozyme

2002

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The class I ligase, a ribozyme previously isolated from random sequence, catalyzes a reaction similar to RNA polymerization, positioning its 5′-nucleotide via a Watson-Crick base pair, forming a 3′,5′-phosphodiester bond between its 5′-nucleotide and the substrate, and releasing pyrophosphate. Like most ribozymes, it requires metal ions for structure and catalysis. Here, we report the ionic requirements of this self-ligating ribozyme. 2+ and Co(NH 3 ) 6 3+ inhibit by binding at least two sites, but they appear to productively fill a subset of the required sites. Inhibition is not the result of a significant structural change, since the ribozyme assumes a nativelike structure when folded in the presence of Ca 2+ or Co(NH 3 ) 6 3+ , as observed by hydroxyl-radical mapping. As further support for a nativelike fold in Ca 2+ , ribozyme that has been prefolded in Ca 2+ can carry out the self-ligation very quickly upon the addition of Mg 2+ . Ligation rates of the prefolded ribozyme were directly measured and proceed at 800 min -1 at pH 9.0.Metal ions are essential for the activity of most ribozymes, functioning in electrostatic shielding, structure, and directly in catalysis (1). Natural ribozymes vary widely in their metal ion specificity, from the large ribozymes (group I intron, group II intron, and RNase P 1 ), which require a divalent metal, preferably Mg 2+ , for activity, to the promiscuous selfcleaving ribozymes (hammerhead, hairpin, VS, and HDV), which are active in many different cations (2-13). Hammerhead, hairpin, and VS ribozymes are even active in nonmetal ions such as NH 4 + (12, 13). Ribozymes selected in vitro also exhibit a wide range of metal ion requirements. Some, like the ribonucleolytic leadzyme, are active only in the cations in which they were selected (14-16). Others exhibit activity in the presence of metal ions which were not present during their selection. For example, an acyl transferase ribozyme, which was selected in the presence of Mg 2+ and K + only, is active in a wide variety of cations, including Co(NH 3 ) 6 3+ (17, 18).The class I ligase, which was also selected in the presence of only Mg 2+ and K + , performs a reaction similar to RNA polymerization, positioning its 5′-nucleotide via a WatsonCrick base pair, forming a 3′,5′-phosphodiester bond between its 5′-nucleotide and the substrate, and releasing pyrophosphate (Figure 1) (19). In fact, a version of this ribozyme can act as a general RNA polymerase, accurately extending a primer up to 14 nucleotides (20). Not only does the ligase perform the same reaction as naturally occurring RNA and DNA polymerases, but the stereospecificity of its preference for sulfur substitutions at the nonbridging oxygens on the R-phosphate matches that of these enzymes, raising the possibility that a catalytic mechanism common to all polymerases extends to ribozyme-mediated polymerization as well (21,22). The ligase is also one of the fastest ribozymes, among both natural ribozymes and those selected in vitro (23). These characteristics r...

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Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer

Cited by 162 publications

References 56 publications

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

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

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

Metal Ion Requirements for Structure and Catalysis of an RNA Ligase Ribozyme

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Tertiary structure formation in the hairpin ribozyme monitored by fluorescence resonance energy transfer

Cited by 162 publications

References 56 publications

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

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

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

Metal Ion Requirements for Structure and Catalysis of an RNA Ligase Ribozyme

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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^,