Direct measurement of oligonucleotide substrate binding to wild-type and mutant ribozymes from Tetrahymena.

Pyle, Anna Marie; McSwiggen, James A.; Cech, Thomas R.

doi:10.1073/pnas.87.21.8187

Cited by 143 publications

(173 citation statements)

References 38 publications

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“…However, binding constants of oligonucleotide substrates and the L-21 ScaI ribozyme determined with nondenaturing polyacrylamide gels are several orders of magnitude larger than predicted simply from base pair formation (Pyle et al, 1990). A similar situation has been described for binding of oligonucleotides to a circular form of the same IVS (Sullivan & Cech, 1985;Sugimoto et al, 1988Sugimoto et al, , 1989.…”

Section: L-21 Sealmentioning

confidence: 67%

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site

Herschlag

Cech²

1990

Biochemistry

317

481

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A ribozyme derived from the intervening sequence (IVS) of the Tetrahymenit preribosomal RNA catalyzes a site-specific endonuclease reaction: G2CCCUCUA5 + G + G2CCCUCU + GAS (G = guanosine). This reaction is analogous to the first step in self-splicing of the pre-rRNA, with the product G2CCCUCU analogous to the 5'-exon. The following mechanistic conclusions have been derived from pre-steady-state and steady-state kinetic measurements at 50 " C and neutral pH in the presence of 10 mM Mg2+. The value of k,,/Km = 9 X lo7 M-' min-' for the oligonucleotide substrate with saturating G represents rate-limiting binding. This rate constant for binding is of the order expected for formation of a RNAaRNA duplex between oligonucleotides. (Phylogenetic and mutational analyses have shown that this substrate is recognized by base pairing to a complementary sequence within the IVS.) The value of k, , = 0.1 min-' represents rate-limiting dissociation of the 5'-exon analogue, G2CCCUCU. The product GAS dissociates first from the ribozyme because of this slow off-rate for G2CCCUCU. The similar binding of the product, G2CCCUCU, and the substrate, G2CCCUCUAS, to the 5'-exon binding site of the ribozyme, with Kd = 1-2 nM, shows that the pAs portion of the substrate makes no net contribution to binding. Both the substrate and product bind -104-fold (6 kcal/mol) stronger than expected from base pairing with the 5'-exon binding site. Thus, tertiary interactions are involved in binding. Binding of G2CCCUCU and binding of G are independent. These and other data suggest that binding of the oligonucleotide substrate, G2CCCUCUA5, and binding of G are essentially random and independent. The rate constant for reaction of the ternary complex is calculated to be k, 350 m i d , a rate constant that is not reflected in the steady-state rate parameters with saturating G. The simplest interpretation is adopted, in which k, represents the rate of the chemical step. A site-specific endonuclease reaction catalyzed by the Tetrahymena ribozyme in the absence of G was observed; the rate of the chemical step with solvent replacing guanosine, k,(-G) = 0.7 m i d , is -500-fold slower than that with saturating guanosine. The value of k,,/Km = 6 X lo7 M-' m i d for this hydrolysis reaction is only slightly smaller than that with saturating guanosine, because the binding of the oligonucleotide substrate is predominantly rate-limiting in both cases. This ribozyme, which approaches the limiting values of k,,/Km for protein enzymes, can be considered to have achieved "catalytic perfection"

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Section: L-21 Sealmentioning

confidence: 67%

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site

Herschlag

Cech²

1990

Biochemistry

317

481

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“…It is currently unclear if this difference reflects the different salt conditions used in obtaining the nearest-neighbor values (1 M NaCl) or effects imposed by the ribozyme structure. Nevertheless, it appears that unlike the Tetrahymena group I ribozyme which binds its product 104-fold tighter than expected from basepairing interactions alone (Herschlag & Cech, 1990;Pyle et al, 1990;Bevilacqua & Turner, 1991), the products of the hammerhead ribozyme motif do not bind considerably stronger then expected from simple helices. P1 may bind somewhat weaker.…”

Section: Discussionmentioning

confidence: 90%

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

1994

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A hammerhead ribozyme (HH16) with eight potential base pairs in each of the substrate recognition helices stabilized product binding sufficiently to enable investigation of the ligation of oligonucleotides bound to the ribozyme. All individual rate constants for product association and dissociation were determined. The following conclusions were obtained for HH16 from the analysis performed at 50 mM Tris, pH 7.5, 10 mM MgC12, and 25 "C.(1) HH16 cleaves bound substrate with a rate constant of k2 = 1 min-l, similar to rate constants obtained with other hammerhead ribozymes. (2) k-2, the rate of ligation of the 5' product and 3' product to form substrate, equaled 0.008 min-I, indicating an approximately 100-fold preference for the formation of products on the ribozyme. This internal equilibrium, compared with that for the overall solution reaction, gives an effective concentration (EC) of M for the two products bound to the ribozyme. This low EC suggests that upon cleavage of S the hammerhead complex acquires a "floppiness" which provides an entropic advantage for the formation of products on the ribozyme. (3) Product and substrate association rate constants were in the range of 107-108 M-1 min-l, comparable to values determined for short helices. (4) The stabilities of ribozyme/product complexes were similar to affinities predicted from helix-coil transitions of simple R N A duplexes, providing no indication of additional tertiary interactions. The products, P1 and P2, stabilize one another 4-fold on the ribozyme. ( 5 ) The dissociation constant for the binding of the substrate to the ribozyme was estimated to be about M.These results allowed the construction of a free energy profile for the reaction of HH16, and provide a basis for future mechanistic studies.Several plant viroids and virusoids autolytically cleave at a specific phosphodiester bond (Buzayan et al., 1986a,b;Hutchins et al., 1986; Prody et al., 1986). A consensus secondary structure of approximately 55 nucleotides termed the "hammerhead" domain has been identified (Buzayan et al., 1986b; Forster & Symons, 1987a,b) which can be assembled from 2 or 3 separate RNA molecules. Thus, the self-cleaving reaction has been turned into a multiple turnover reaction, with separate oligonucleotides acting as the "ribozyme" and the "substrate" (Figure 1) (Sampson et al., 1987;Uhlenbeck, 1987;Haseloff & Gerlach, 1988;Koizumi et al., 1988 Koizumi et al., , 1989 Jeffries & Symons, 1989 p3WAACGUC>p; PI-G, GGGAACGUCG; P1-3'p, GGGAACGUC3'-p; P2, GUCGUCGC; P2-C, GUCGUCGCC; P2-p'zCp, GUCGUCGCp"Cp; S-C, GG-G AACGUCGUCGUCGCC; p3*S-C, p3WAACGUCGUCGUCGCC; S-p'zCp, GGGAACGUCGUCGUCGCpWp; S, GGGAACGUC-GUCGUCGC; p32S, p32GGGAACGUCGUCGUCGC; E, ribozyme; HH16, hammerhead cleavage motif comprised of separate ribozyme and substrate oligonucleotides ( (HH 16). Using standardized hammerhead nomenclature (Hertel et al., 1992), the ribozyme (E), comprised of 38 nucleotides, catalyzes thecleavageof a specific phosphodiester bond within its 1 8-nucleotidelong substrate (S-C), ...

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“…A 10-fold excess of the chase solution containing unlabeled rP in reaction buffer at 50°C was then added, and aliquots were removed at specified times. The aliquots were immediately added to a loading buffer containing 40% glycerol and 0.5% xylene cyanol and carefully loaded onto a 10% native polyacrylamide gel while the gel was running (Pyle et al, 1990). The gel running buffer contained 100 mM Tris-HEPES (34 mM Tris and 66 mM HEPES, pH 7.5), 0.1 mM EDTA, and 10 mM MgCl 2 , and the running temperature was maintained at 10°C to limit further dissociation.…”

Section: Methodsmentioning

confidence: 99%

Mechanistic Investigations of a Ribozyme Derived from the Tetrahymena Group I Intron: Insights into Catalysis and the Second Step of Self-Splicing

Mei

Herschlag

1996

Biochemistry

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Self-splicing of Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps. One major difference between these steps is that in the first an exogenous guanosine (G) binds to the active site, while in the second the 3′-terminal G414 residue of the intron binds. The first step has been extensively characterized in studies of the L-21Scal ribozyme, which uses exogenous G as a nucleophile. In this study, mechanistic features involved in the second step are investigated by using the L-21G414 ribozyme. The L-21G414 reaction has been studied in both directions, with G414 acting as a leaving group in the second step and a nucleophile in its reverse. The rate constant of chemical step is the same with exogenous G bound to the L-21ScaI ribozyme and with the intramolecular guanosine residue of the L-21G414 ribozyme. The result supports the previously proposed single G-binding site model and further suggests that the orientation of the bound G and the overall active site structure is the same in both steps of the splicing reaction. An evolutionary rationale for the use of exogenous G in the first step is also presented. The results suggest that the L-21G414 ribozyme exists predominantly with the 3′-terminal G414 docked into the G-binding site. This docking is destabilized by ∼100-fold when G414 is attached to an electron-withdrawing pA group. The internal equilibrium with K int ) 0.7 for the ribozyme reaction indicates that bound substrate and product are thermodynamically matched and is consistent with a degree of symmetry within the active site. These observations are consistent with the presence of a second Mg ion in the active site. Finally, the slow dissociation of a 5′ exon analog relative to a ligated exon analog from the L-21G414 ribozyme suggests a kinetic mechanism for ensuring efficient ligation of exons and raises new questions about the overall self-splicing reaction.Self-splicing of the Tetrahymena pre-rRNA proceeds in two consecutive phosphoryl transesterification steps ( Figure 1A; Cech, 1990). In the first step, an exogenous guanosine (G) 1 acting as a nucleophile attacks the 5′ splice site, leaving the 5′ exon with a 3′-hydroxyl terminus. In the second step, the 3′-terminal residue of the intron, G414, binds to the active site, and its 3′ phosphoryl group is attacked by the 3′-hydroxyl of the 5′ exon. This gives ligated exons and the free intron.The first step of self-splicing has been studied by using a shortened version of the intron. This version, referred to as the L-21ScaI ribozyme ( Figure 2B), has the first 21 and last 5 nucleotides removed and mimics the first step of the selfsplicing reaction ( Figure 1B). Extensive studies of the L-21ScaI ribozyme have provided insights into the mechanism of RNA catalysis and the first step of self-splicing (Cech et al., 1992).The second step of self-splicing, however, has not been characterized in as much detail. Several components have been shown to be involved in this step (Figure 2A). Guanosine is universally conserved a...

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Direct measurement of oligonucleotide substrate binding to wild-type and mutant ribozymes from Tetrahymena.

Cited by 143 publications

References 38 publications

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site

A Kinetic and Thermodynamic Framework for the Hammerhead Ribozyme Reaction

Mechanistic Investigations of a Ribozyme Derived from the Tetrahymena Group I Intron: Insights into Catalysis and the Second Step of Self-Splicing

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