Kinetic Mechanism of the Hairpin Ribozyme

Abstract

To investigate the relationship between RNA folding and ribozyme catalysis, we have carried out a detailed kinetic analysis of four structural derivatives of the hairpin ribozyme. Optimal and suboptimal (wild-type) substrate sequences were studied in conjunction with stabilization of helix 4, which supports formation of the catalytic core. Pre-steady-state and steady-state kinetic studies strongly support a model in which each of the ribozyme variants partitions between two major conformations leading to active and inactive ribozyme· substrate complexes. Reaction rates for cleavage, ligation, and substrate binding to both ribozyme conformations were determined. Ligation rates (3 min−1) were typically 15-fold greater than cleavage rates (0.2 min−1), demonstrating that the hairpin ribozyme is an efficient RNA ligase. On the other hand, substrate binding is very rapid (kon = 4 × 108m−1 min−1), and the ribozyme· substrate complex is very stable (KD< 25 pm; koff < 0.01 min−1). Stabilization of helix 4 increases the proportion of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates. These effects can be explained by stabilization of the catalytic core of the ribozyme. Rigorous consideration of conformational isomers and their intrinsic kinetic properties was necessary for development of a kinetic scheme for the ribozyme-catalyzed reaction. To investigate the relationship between RNA folding and ribozyme catalysis, we have carried out a detailed kinetic analysis of four structural derivatives of the hairpin ribozyme. Optimal and suboptimal (wild-type) substrate sequences were studied in conjunction with stabilization of helix 4, which supports formation of the catalytic core. Pre-steady-state and steady-state kinetic studies strongly support a model in which each of the ribozyme variants partitions between two major conformations leading to active and inactive ribozyme· substrate complexes. Reaction rates for cleavage, ligation, and substrate binding to both ribozyme conformations were determined. Ligation rates (3 min−1) were typically 15-fold greater than cleavage rates (0.2 min−1), demonstrating that the hairpin ribozyme is an efficient RNA ligase. On the other hand, substrate binding is very rapid (kon = 4 × 108m−1 min−1), and the ribozyme· substrate complex is very stable (KD< 25 pm; koff < 0.01 min−1). Stabilization of helix 4 increases the proportion of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates. These effects can be explained by stabilization of the catalytic core of the ribozyme. Rigorous consideration of conformational isomers and their intrinsic kinetic properties was necessary for development of a kinetic scheme for the ribozyme-catalyzed reaction. Since the first description of a catalytically active RNA molecule (1Cech T.R. Zaug A.J. Grabowski P.J. Cell. 1981; 27: 487-496Google Scholar), much effort has been focused toward elucidating the molecular mechanisms of ribozyme catalysis. Valuable information has emerged from detailed kinetic and thermodynamic analyses of intramolecular and intermolecular reactions catalyzed by several naturally occurring ribozymes, including self-splicing group I introns (2Herschlag D. Cech T.R. Biochemistry. 1990; 29: 10159-10171Google Scholar, 3Bevilacqua P.C. Kierkez R. Johnson K.A. Turner D.H. Science. 1992; 258: 1355-1358Google Scholar, 4Bevilacqua P.C. Sugimoto N. Turner D.H. Biochemistry. 1996; 35: 648-658Google Scholar, 5Golden B.L. Cech T.R. Biochemistry. 1996; 35: 3754-3763Google Scholar, 6Mei R. Herschlag D. Biochemistry. 1996; 35: 5796-5809Google Scholar) and group II introns (7Pyle A.M. Green J.B. Biochemistry. 1994; 33: 2716-2725Google Scholar, 8Michels Jr., W.J. Pyle A.M. Biochemistry. 1995; 34: 2965-2977Google Scholar, 9Daniels D.L. Michels Jr., W.J. Pyle A.M. J. Mol. Biol. 1996; 256: 31-49Google Scholar), ribonuclease P (10Beebe J.A. Fierke C.A. Biochemistry. 1994; 33: 10294-10304Google Scholar, 11Beebe J.A. Kurz J.C. Fierke C.A. Biochemistry. 1996; 35: 10493-10505Google Scholar), hammerhead ribozymes (12Hertel K.J. Herschlag D. Uhlenbeck O.C. Biochemistry. 1994; 33: 3374-3385Google Scholar, 13Hertel K.J. Uhlenbeck O.C. Biochemistry. 1995; 34: 1744-1749Google Scholar). and hairpin ribozymes (14Hegg L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google Scholar). It is widely accepted that the folded structure of RNA is critical for its catalytic activity. However, few studies have addressed the problem of how differences in ribozyme folding may affect individual steps of the reaction pathway. One major complication in kinetic analysis of ribozymes results from the ability of most RNA molecules to fold into multiple conformations (15Uhlenbeck O.C. RNA. 1995; 1: 4-6Google Scholar). We believe that the study of conformationally heterogeneous ribozymes is important because it represents a direct and realistic approach to the problem of RNA structure and function. As a model to investigate the relationship between RNA structure and kinetic behavior, we are studying the hairpin ribozyme. This ribozyme is a relatively small RNA molecule (50 nucleotides, 17 kDa) derived from the minus strand of the satellite RNA associated with tobacco ringspot virus (16Feldstein P.A. Buzayan Scholar, R. Biochemistry. Scholar, J. Scholar). It a RNA a a to and molecules with structure of the ribozyme· substrate the and for catalysis, has been and in Mol. Biol. 1994; Scholar). substrate with the ribozyme two intermolecular and by a of four in both substrate and ribozyme. the two intramolecular and are by a for are and and have to the that and for the ribozyme catalytic core to be a between helix and helix Mol. Biol. 1996; Scholar). This has support from which that can be of the and J. Biol. 1995; Scholar). and its an folding by and studies J. Mol. Biol. 1994; Scholar, Biochemistry. 1994; 33: Scholar). to a first the of the is for ribozyme J. Scholar), we that of helix 4 a in catalytic the active structure of by Biochemistry. 1995; 34: Scholar). To a of in the hairpin ribozyme we have carried out a detailed kinetic analysis of structural variants of ribozyme. the of helix 4 was its is to affect the of the catalytic core. hairpin ribozymes with substrate were because the naturally occurring substrate is conformationally heterogeneous Biochemistry. Scholar). four derivatives of the hairpin ribozyme that two were As in the helix 4 was with and a stable and the of the substrate and ribozyme were to substrate in J. Biol. 1995; Scholar). four hairpin ribozymes were in with their be helix helix and the of an helix 4 be to This analysis has the relationship between RNA folding and by two conformational of the hairpin ribozyme and the in of individual These results important into folding of the hairpin ribozyme and how structural can be in kinetic We that results be for the of ribozymes with folding and catalytic for ribozyme and ribozyme were an and RNA of the to the RNA substrate was were by with RNA Uhlenbeck O.C. Scholar). and RNA molecules were by Biochemistry. 1995; 34: Scholar). RNA of were by RNA were with and RNA in ribozymes were with reactions were carried out in a reaction and 25 and substrate were for in reaction of ribozymes was to formation of ribozyme Biochemistry. 1994; 33: Scholar). were to for 25 were by of the ribozyme and of the reaction were and with an of were in were a reactions were carried out with ribozyme and than in the in the of cleavage was ribozyme that ribozyme is of substrate was and to and for the and the of the and the of the and for the rates. represents the of the cleavage reaction and was typically between and These were by analysis the and for the was typically than from was than for ribozymes and than for carried out by were than It is important to that the of a which reactions that were with a the was for reactions were carried out with substrate and ribozyme. were to for each substrate substrate were to the of the formation reaction to were by the to the for substrate steady-state were by analysis reactions in ligation molecules in which the of the substrate is a to the of the ribozyme 1992; Scholar). These molecules were by from in the of RNA and the to the cleavage a was Biochemistry. 1995; 34: Scholar). cleavage was by Ligation reactions were carried out with ribozymes cleavage and cleavage the the of ligation was by the of the cleavage that is to of ribozyme was and to the of ligation was typically between and were by of analysis and were for the cleavage reaction. cleavage with a was from a reaction cleavage was Biochemistry. 1995; 34: Scholar). Ligation reactions were carried out with a small of cleavage than and of cleavage and ribozyme of cleavage to the cleavage was and to the and rates of the were for the cleavage reaction. small of than was with a of ribozyme in the reaction for 25 These formation of the complex the binding are and for the and the ribozymes, is by a of a that is to the substrate a substrate for reactions carried out with ribozymes, was of the RNA substrate because molecule stable the were and with an of were and reactions were carried out in the of the of the was by the substrate with the molecule the the RNA to the of ribozyme. cleavage of the substrate was that is of the substrate the carried out in the of the molecule were to reactions in the of and were of the kinetic and was carried out for substrate association were a of to to substrate ribozyme from to ribozymes from to ribozymes, were with a than of the in reaction 25 each ribozyme several reactions were from to 4 molecule was a in the of the RNA in the of the of the molecule was for reactions carried out with were for 25 of the This is to a cleavage of the for the cleavage reaction is of the cleavage reaction were to and the rates were ribozyme to association and rates (2Herschlag D. Cech T.R. Biochemistry. 1990; 29: 10159-10171Google Scholar). the four ribozymes and with we have carried out and steady-state kinetic Pre-steady-state were to individual rates for substrate binding and and and analysis was to steps and to the rates between conformations of the ribozyme in which ribozyme was in substrate were to the for cleavage of substrate and the the of the cleavage both cleavage to the ribozyme to be However, is much than ligation in the hairpin ribozyme (14Hegg L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google Scholar), the of the ligation in a cleavage carried out in the of ribozyme for cleavage catalyzed by and ribozymes were 25 of was with of their to cleavage rates and for a cleavage catalyzed by the ribozymes is in four ribozymes, the were to and and As in the are much than This was by of the and from the of and I the of the rates and for the two reaction for four the of we to and to their rates. and rates of both the were of ribozyme This out the that the of the ribozymes is to the of two of the both the ribozyme and the substrate were for To folding were the cleavage reactions were These for for for and were for the cleavage is in the and and to the two of the and to the two of the were to the of were to the of reactions were carried out and and rates were by the to and from were typically than for the of the ribozymes and than for of the and to the two of the is in the and were to the of in a reactions were carried out and and rates were by the to and from were typically than for the of the ribozymes and than for of the in the of the of the substrate was in the of the reaction. of the was greater than the of the the reaction rates a for the the of the and were This that the of the two in the of the ribozyme is from the other variants of the hairpin ribozyme. rates of ligation for four of the hairpin ribozyme were two and was carried out a of the hairpin ribozyme that is to the a 1992; Scholar), and was a of the ribozyme with a of the reaction was a of in the of of ribozyme and and both the ligation an approach to the between cleavage and ligation cleavage is much than of the complex the be the of cleavage and ligation rates. were studied for each of the four ribozymes, and reaction 4 and On the other hand, the ligation reaction in catalyzed by and ribozymes and These ligation were to which of and rates of both of for and ribozymes be the ribozyme for reactions to of ribozyme Biochemistry. 1994; 33: for the ligation is in the and reactions were carried out and a of to the cleavage a in the of of the cleavage carried out were by the to and reactions were carried out with a of cleavage in the of of both cleavage and ribozyme. and the two of the and rates were by the to and from were typically than and the two of the and rates were by the to and from were typically than were to the of were to the of reactions were carried out and a of to the cleavage a in the of of the cleavage carried out were by the to and reactions were carried out with a of cleavage in the of of both cleavage and ribozyme. and the two of the and rates were by the to and from were typically than is in the and were to the of in a ligation rates were 15-fold than the cleavage rates I with the rates in the ligation the reaction is This that the hairpin ribozyme is an efficient ligase. was from a kinetic study with the hairpin ribozyme (14Hegg L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google Scholar). Ligation rates were for the with with the of reactions carried out with the ribozymes were to for substrate rates. As in the of a complex between substrate and cleavage ribozyme· substrate complex is by a of substrate with an of ribozyme This is to binding of substrate for substrate association that most of the substrate is by an of a to the substrate an of substrate and of substrate is the of the of the between cleavage and substrate These were carried out with the four ribozymes study and with ribozyme is in reaction the However, reactions carried out in the of the for and rates were by the to with are in and that the in the of with the of the into both and rates. of substrate molecules is the is the This was with ribozymes a of for and rates for a cleavage reaction to substrate and the two of the in the of the reaction. was for the its for from were typically than and the two of the in the of the reaction. was for the its for from were typically than were to a direct of the and were to a direct of the and reactions in which molecule was were with formation of substrate ribozyme reactions were the ribozyme and reactions in which molecule was were with formation of substrate ribozyme and the two of the in the of the reaction. was for the its for from were typically than were to a direct of the and in a reactions were the ribozyme and These results that are two of complexes. One cleavage a much than substrate and is for the of the kinetic the other than cleavage and is for the in cleavage kinetic that into two be results in the to the association for is in of substrate was with an of ribozyme for to formation of the two of of a to the substrate an of substrate was to binding were to was and As of ribozyme· substrate cleavage the substrate in the other into the of substrate the represents the formation of the catalytically complex the of complex formation of ribozyme are in rates of complex formation the of approach to which is the of association and rates and koff for association and and represents ribozyme of ribozyme is in was to that substrate is very substrate association rates for the four ribozymes study are in of substrate association rates is because of the that two are As the for that is in can be a of the substrate association rates of both of substrate association is in the and × × association rates were by ribozyme and is in the and in a association rates were by ribozyme and To the of of the kinetic from the we carried out cleavage reactions steady-state in which the is This analysis is two of are rates between may be in the cleavage reactions were carried out with and ribozymes and steady-state were with with ribozymes the substrate by of the for both and ribozymes, the for formation substrate was and the substrate which of the is was of formation for to substrate and of formation was These that the ribozyme is into an inactive and to to the kinetic of studies of ribozyme the of folded of ribozymes and is a problem that an kinetic description of the reaction in most studies to have problem by of ribozymes and and that are and in the have focused the of the reaction by rates of reaction. hairpin ribozyme to the that conformational was to be an of the hairpin ribozyme. an of the kinetic consideration of the kinetic that is We have individual for substrate binding and for four structural variants of the hairpin ribozyme in an effort to the structural for conformational with its for the catalytic of the ribozyme. It is important to out that each of four ribozymes in However, two major and were for of kinetic the we two conformations in of is stable the it is that be with a for the conformational of RNA. of the cleavage reactions catalyzed by the four hairpin ribozymes and is very have been in other ribozyme reactions B.L. Cech T.R. Biochemistry. 1996; 35: 3754-3763Google Scholar, 9Daniels D.L. Michels Jr., W.J. Pyle A.M. J. Mol. Biol. 1996; 256: 31-49Google Scholar, Biochemistry. Scholar, P.J. J. Mol. Biol. 1996; Scholar, P.J. Biochemistry. 1994; 33: Scholar, J. Biochemistry. 1996; 35: Scholar). However, in a very few the of the reactions was into the analysis of the B.L. Cech T.R. Biochemistry. 1996; 35: 3754-3763Google Scholar, 9Daniels D.L. Michels Jr., W.J. Pyle A.M. J. Mol. Biol. 1996; 256: 31-49Google Scholar, J. Biochemistry. 1996; 35: Scholar). were ribozyme and with for RNA folding to reaction. to an of the hairpin ribozyme. kinetic mechanisms to cleavage reactions and heterogeneous of of the is necessary for the of rates for cleavage, and ligation the of the hairpin the of the cleavage reaction is substrate is and the that for results the of two of complexes. from the cleavage in of the the of cleavage be much than the of substrate the the of the is substrate is other of than substrate molecules be to between both ribozyme is in in of the from the substrate in the between cleavage and the be the active in of the an inactive a model has been it is to information from the and rates of the to the model the of active and inactive substrate binding to the of the and cleavage can be from the of the of the As the from the active complex has to be much than the cleavage the can be to be min−1). This is in with kinetic studies of the hairpin ribozyme (14Hegg L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google RNA. Scholar). On the other hand, the of the inactive is to the of the substrate is to between active and inactive each and substrate molecules that are to the active to the of the reaction. the is the proportion of the of the to the substrate that partitions to active which is the of the = of of to the in can be 0.01 from to information the rates between the two can be from the in that the of the reaction substrate is that substrate from the inactive complex than to active other substrate is to be than a an of can be To information the kinetic scheme has to be with As in active and inactive of can be a substrate molecule is to the ribozyme in two conformations two of ribozymes are to substrate binding These two for a cleavage reaction carried out in steady-state that with an of substrate ribozyme. to the ribozyme molecules into active and inactive each cleavage the of active with to a in the of formation the ribozyme into inactive complexes. It is important to that a between both be to the of active and inactive the = min−1) is much than the for the of inactive into active < min−1). However, the steady-state of formation was for to that the model in is On the the kinetic scheme in is with of from both and steady-state to the hairpin ribozyme is to two substrate binding to both active and inactive are active to substrate the inactive to model in and into the results from the steady-state it is to an for the from active to inactive the can that a a in the steady-state of formation the of active complex is that the steady-state of cleavage reaction and an of it can be be than and into active and inactive to be in very model in a of the and substrate association rates. to active and inactive ribozyme with association rates to the complexes. into that substrate very in both it can be that the of complex formation is the for both the of their association rates and by the of each we the ribozyme in the active represents the of active and inactive the of complex formation for both is 4 the association rates can be the for the formation of the active and inactive complexes. On the other hand, the of each complex the substrate has been that is the of the and can be derived the of each is by both the of ribozyme in each and its substrate association to it is very important to the of ribozyme in the kinetic analysis the association rates and the of complex formation be by their of is by the results with the steady-state a of the cleavage (14Hegg L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google Scholar) and a between active and inactive the steady-state and can be with the individual in that substrate is much than cleavage and can be to the to the results of the multiple cleavage reactions the for be This is in with from 4 and the of the and the association and for and ribozymes, It is important to that the of in are the of substrate from both active and inactive is much than the cleavage for the including the in is the of the model in has been by of for the rates of substrate and results from both and steady-state were by of an kinetic in is necessary for a of the for the hairpin ribozyme One of the first that can be from the kinetic analysis in is the suboptimal cleavage of the ribozyme. into the of its and it can be that of the are in the active On the the other ribozymes and active in of the two structural of helix 4 and of substrate are to the catalytic of the hairpin ribozyme. However, the effects of two to be the of substrate of helix 4 the of active and inactive in the of the cleavage reaction. into the kinetic of the association rates and 4, results that of helix 4 have in ribozyme folding the substrate is was in the of substrate the of active was by helix As in can be explained by a substrate association for the active by an in the of folded ribozymes two are we out the that may be the results in that the conformational of the hairpin ribozyme is toward the active the of helix 4 is This is by studies Biochemistry. 1995; 34: Scholar). Stabilization of helix 4 may the of in the catalytic core of the ribozyme. of helix has been for the virus ribozyme Biochemistry. 1996; 35: Scholar). However, the of the substrate is It is that the substrate binding strand is with the folding of the ribozyme. the substrate helix 4 the that a ribozyme molecule the active It is that the hairpin ribozymes helix 4, substrate in a cleavage have been in a kinetic analysis of the hairpin ribozyme by and Fedor (14Hegg L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google Scholar). It is important to that be substrate from the inactive is than the cleavage the and Fedor results are with a model substrate from the inactive was relatively in their This is by the of the that of the substrate the of a (14Hegg L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google Scholar). we that conformational is a of the hairpin ribozyme and that it structural of ribozyme. results in information the structure of the inactive However, structural analyses carried out in support the of an inactive of the complex in which of and results in an that of the and in inactive has been for a hairpin ribozyme Biochemistry. 1996; 35: Scholar). of substrate association rates an important between the and of formation of the complex is than that of the of the of helix 4 are However, the association rates with the substrate were to the for helix formation between D. J. Mol. Biol. Scholar, J. Mol. Biol. Scholar, D. Uhlenbeck O.C. Scholar, J. J. 1: Scholar, K.J. Scholar, Jr., Biochemistry. Scholar). association of the substrate is to be to its ability to hairpin and conformations by of that the intrinsic association is by substrate the for the substrate that most of the molecules are in a that is for ribozyme On the other hand, the of the substrate to be in rapid the of substrate association the ribozyme This is in with the rates for model and of J. J. Mol. Biol. Scholar). It be that the for the substrate association of the hairpin ribozyme was with a substrate (14Hegg L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google Scholar), and that is to the we for the association the that the binding of a complex was to that of a ribozyme to the cleavage (14Hegg L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google Scholar). However, we the association of a substrate to be the for substrate binding is than that for binding of the cleavage the complex may be from the complex by the ribozyme with the cleavage ligation reaction catalyzed by the hairpin ribozyme the Ligation reactions carried out in that the cleavage is to the On the other hand, a reaction was the ribozyme was to of the cleavage This is in with the model for the cleavage reaction. between the ribozyme and the cleavage to the of the its to the ligation be the of the of an inactive of complexes. a of the ribozyme is It is that the of the from of inactive was much for the ligation reactions than for the cleavage This is to the greater of the with that of the cleavage (14Hegg L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google Scholar). of the ligation rates in were 15-fold greater than the cleavage rates. These results with the that the hairpin ribozyme is efficient than an (14Hegg L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google Scholar). We that ligation rates in most by helix were the ligation reactions carried out in the were than the cleavage and was between and It has been that the of the ligation reaction of the hairpin ribozyme results from a ribozyme which the of the cleavage reaction (12Hertel K.J. Herschlag D. Uhlenbeck O.C. Biochemistry. 1994; 33: 3374-3385Google Scholar, L.A. Fedor M.J. Biochemistry. 1995; 34: 15813-15828Google Scholar). to the ligation helix 4 was may a structure of the hairpin ribozyme helix is with This with the of substrate association for and It has been that helix formation is a of a of an of two to and a in which the of to the the helix D. J. Mol. Biol. Scholar, J. Mol. Biol. Scholar). the structure by the of helix 4 the formation of the the for the of the results in that of ribozyme structure may have effects catalytic of the kinetic of the including the kinetic of conformational is an important for the cleavage is carried out substrate it can be that both ribozyme are in the of complex the cleavage reaction is by and binding of a substrate the between active and inactive conformations of the ribozyme can be to the of the active can be derived from reaction = the of active complexes. for can be by that the of active of cleavage reaction carried out with the ribozyme in steady-state This was by of an = an of and of its have been a in the of formation in steady-state Since substrate is much than the association rates in the of substrate binding can be to the reaction scheme and into that both ribozyme and are in substrate the of each complex and can be for the of active ribozyme = of each complex can be from 17 We for of RNA for the of and RNA and and for