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D Herschlag

Publications and source records attributed to D Herschlag.

At least 55 records · Page 3Linked to original sources
PubMed1998

The DEAD box protein eIF4A. 2. A cycle of nucleotide and RNA-dependent conformational changes.

Limited proteolysis experiments have been carried out with the DEAD box protein eIF4A. The results suggest that there is a substantial conformational change in eIF4A upon binding single-stranded RNA. Binding of ADP induces conformational changes in the free enzyme and the enzyme.RNA complex, and binding of the ATP analogue AMP-PNP induces a conformational change in the enzyme.RNA complex. The presence or absence of the gamma-phosphate on the bound nucleotide acts as a switch, presumably via the Walker motifs, that mediates changes in protein conformation and, as described in the preceding paper in this issue, also mediates changes in RNA affinity. Thus, these results suggest that there is a series of changes in conformation and substrate affinity throughout the ATP hydrolysis reaction cycle. A model is proposed in which eIF4A and the eIF4A-like domains of the DEAD box proteins act as ATP-driven conformational switches or motors that produce movements or structural rearrangements of attached protein domains or associated proteins. These movements could then be used to rearrange RNA structures or RNA.protein complexes.

Adenine Nucleotides↗
PubMed1998

Structure-function relationships in the hammerhead ribozyme probed by base rescue.

We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.

Adenine↗
PubMed1997

Involvement of a specific metal ion in the transition of the hammerhead ribozyme to its catalytic conformation.

Previous crystallographic and biochemical studies of the hammerhead ribozyme suggest that a metal ion is ligated by the pro-Rp oxygen of phosphate 9 and by N7 of G10.1 and has a functional role in the cleavage reaction. We have tested this model by examining the cleavage properties of a hammerhead containing a unique phosphorothioate at position 9. The Rp-, but not Sp-, phosphorothioate reduces the cleavage rate by 10(3)-fold, and the rate can be fully restored by addition of low concentrations of Cd2+, a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite its location approximately 20 A from the cleavage site in the crystal structure. Analysis of the concentration dependence suggests that Cd2+ binds with a Kd of 25 microM in the ground state and a Kd of 2.5 nM in the transition state. The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand in the transition state and that this metal ion may participate in a large scale conformational change that precedes hammerhead cleavage.

Base Sequence↗
PubMed1997

Use of intrinsic binding energy for catalysis by an RNA enzyme.

The contribution of several individual ribozyme.substrate base pairs to binding and catalysis has been investigated using hammerhead ribozyme substrates that were truncated at their 3' or 5' ends. The base pairs at positions 1.1-2.1 and 15.2-16.2, which flank the conserved core, each contribute 10(4)-fold in the chemical step, without affecting substrate binding. In contrast, base pairs distal to the core contribute to substrate binding but have no effect on the chemical step. These results suggest a "fraying model" in which each ribozyme.substrate helix can exist in either an unpaired ("open") state or a helical ("closed") state, with the closed state required for catalysis. The base pairs directly adjacent to the conserved core contribute to catalysis by allowing the closed state to form. Once the number of base pairs is sufficient to ensure that the closed helical state predominates, additional residues provide stabilization of the helix, and therefore increase binding, but have no further effect on the chemical step. Remarkably, the >5 kcal/mol free energy contribution to catalysis from each of the internal base pairs is considerably greater than the free energy expected for formation of a base pair. It is suggested that this unusually large energetic contribution arises because free energy that is typically lost in constraining residues within a base pair is expressed in the transition state, where it is used for positioning. This extends the concept of "intrinsic binding energy" from protein to RNA enzymes, suggesting that intrinsic binding energy is a fundamental feature of biological catalysis.

Catalysis↗
PubMed1997

Effects of divalent metal ions on individual steps of the Tetrahymena ribozyme reaction.

The Tetrahymena thermophila L-21 ScaI ribozyme utilizes Mg2+ to catalyze a site-specific endonuclease reaction analogous to the first step of self-splicing. To better understand the contribution of Mg2+ to ribozyme activity, the Mg2+ concentration dependence of individual rate constants was examined at concentrations greater than those required for ribozyme folding (>2 mM; at 50 degrees C and pH 6.7). Analysis of metal ion inhibition of the chemical step of the reaction indicated that two Ca2+ ions compete with two Mg2+ ions involved in active site chemistry. These Mg2+ ions are bound tightly to the E.S complex (Kd < 2 mM). The rate constant for association of the oligoribonucleotide substrate (S) increased 12-fold from 2 to 100 mM Mg2+ and exhibited saturation behavior, consistent with a single Mg2+ ion involved in S association that binds to the free ribozyme with a Kd for Mg2+ of 15 mM. The preference for the divalent metal ion (Mg2+ congruent with Ca2+ > Ba2+ >> Sr2+) suggested that enhancing the rate constant of S association is not simply a function of ionic strength, but is due to a distinct metal ion binding site. Even though Ca2+ does not support reaction, the RNA substrate S was able to bind in the presence of Ca2+. Upon addition of Mg2+, S was cleaved without first dissociating. A model is proposed in which the inactive Ca2+ form of E.S is structurally equivalent to the open complex along the reaction pathway, which has the RNA substrate bound but not docked into the active site. Weaker binding of S in Ca2+ was shown to result from an increase in the rate constant of S dissociation, leading to the proposal that a tight Mg2+ binding site or sites in the E.S complex contribute to the strong binding of S. In summary, the data provide evidence for four functions for bound Mg2+ ions in the catalytic cycle: one increases the rate of RNA substrate binding, one or more decrease the rate of dissociation of S, and two are involved in the chemical step.

Animals↗
PubMed1997

Quantitating tertiary binding energies of 2' OH groups on the P1 duplex of the Tetrahymena ribozyme: intrinsic binding energy in an RNA enzyme.

Binding of the Tetrahymena ribozyme's oligonucleotide substrate (S) involves P1 duplex formation with the ribozyme's internal guide sequence (IGS) to give an open complex, followed by docking of the P1 duplex into the catalytic core via tertiary interactions to give a closed complex. The overall binding energies provided by 2' OH groups on S and IGS have been measured previously. To obtain the energetic contribution of each of these 2' OH groups in the docking step, we have separately measured their contribution to the stability of a model P1 duplex using "substrate inhibition". This new approach allows measurement of duplex stabilities under conditions identical to those used for ribozyme binding measurements. The tertiary binding energies from the individual 2' OH groups include a small destabilizing contribution of 0.7 kcal/mol and stabilizing contributions of up to -2.9 kcal/mol. The energetic contributions of specific 2' OH groups are discussed in the context of considerable previous work that has characterized the tertiary interactions of the P1 duplex. A "threshold" model for the open and closed complexes is presented that provides a framework to interpret the energetic effects of functional group substitutions on the P1 duplex. The sum of the tertiary stabilization provided by the conserved G x U wobble at the cleavage site and the individual 2' OH groups on the P1 duplex is significantly greater than the observed tertiary stabilization of S (11.0 vs 2.2 kcal/mol). It is suggested that there is an energetic cost for docking the P1 duplex into the active site that is paid for by the "intrinsic binding energy" of groups on the P1 duplex. Substrates that lack sufficient tertiary binding energy to overcome this energetic barrier exhibit reduced reactivities. Thus, the ribozyme appears to use the intrinsic binding energy of groups on the P1 duplex for catalysis. This intrinsic binding energy may be used to position reactants within the active site and to induce electrostatic destabilization of the substrate, relative to its interactions in solution.

Animals↗
PubMed1997

Mechanistic aspects of enzymatic catalysis: lessons from comparison of RNA and protein enzymes.

A classic approach in biology, both organismal and cellular, is to compare morphologies in order to glean structural and functional commonalities. The comparative approach has also proven valuable on a molecular level. For example, phylogenetic comparisons of RNA sequences have led to determination of conserved secondary and even tertiary structures, and comparisons of protein structures have led to classifications of families of protein folds. Here we take this approach in a mechanistic direction, comparing protein and RNA enzymes. The aim of comparing RNA and protein enzymes is to learn about fundamental physical and chemical principles of biological catalysis. The more recently discovered RNA enzymes, or ribozymes, provide a distinct perspective on long-standing questions of biological catalysis. The differences described in this review have taught us about the aspects of RNA and proteins that are distinct, whereas the common features have helped us to understand the aspects that are fundamental to biological catalysis. This has allowed the framework that was put forth by Jencks for protein catalysts over 20 years ago (1) to be extended to RNA enzymes, generalized, and strengthened.

Animals↗
PubMed1996

The change in hydrogen bond strength accompanying charge rearrangement: implications for enzymatic catalysis.

The equilibrium for formation of the intramolecular hydrogen bond (KHB) in a series of substituted salicylate monoanions was investigated as a function of delta pKa, the difference between the pKa values of the hydrogen bond donor and acceptor, in both water and dimethyl sulfoxide. The dependence of log KHB upon delta pKa is linear in both solvents, but is steeper in dimethyl sulfoxide (slope = 0.73) than in water (slope = 0.05). Thus, hydrogen bond strength can undergo substantially larger increases in nonaqueous media than aqueous solutions as the charge density on the donor or acceptor atom increases. These results support a general mechanism for enzymatic catalysis, in which hydrogen bonding to a substrate is strengthened as charge rearranges in going from the ground state to the transition state; the strengthening of the hydrogen bond would be greater in a nonaqueous enzymatic active site than in water, thus providing a rate enhancement for an enzymatic reaction relative to the solution reaction. We suggest that binding energy of an enzyme is used to fix the substrate in the low-dielectric active site, where the strengthening of the hydrogen bond in the course of a reaction is increased.

Dimethyl Sulfoxide↗
PubMed1996

Rescue of abasic hammerhead ribozymes by exogenous addition of specific bases.

We have synthesized 13 hammerhead ribozyme variants, each containing an abasic residue at a specific position of the catalytic core. The activity of each of the variants is significantly reduced. In four cases, however, activity can be rescued by exogenous addition of the missing base. For one variant, the rescue is 300-fold; for another, the rescue is to the wild-type level. This latter abasic variant (G10.1X) has been characterized in detail. Activation is specific for guanine, the base initially removed. In addition, the specificity for guanine versus adenine is substantially altered by replacing C with U in the opposite strand of the ribozyme. These results show that a binding site for a small, noncharged ligand can be created in a preexisting ribozyme structure. This has implications for structure-function analysis of RNA, and leads to speculations about evolution in an "RNA world" and about the potential therapeutic use of ribozymes.

2-Aminopurine↗
PubMed1996

Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach to this problem: the uncatalyzed hydrolysis of GTP is analyzed, and the understanding derived is applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, this analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl (-PO(3)2-) transfer, including kinases and phosphatases.

Amino Acid Sequence↗
PubMed1996

Specificity of hammerhead ribozyme cleavage.

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths.

Base Sequence↗
PubMed1996

Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: insights into catalysis and the second step of self-splicing.

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-21ScaI 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 approximately 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.

Animals↗
PubMed1996

The energetics of hydrogen bonds in model systems: implications for enzymatic catalysis.

Low-barrier or short, strong hydrogen bonds have been proposed to contribute 10 to 20 kilocalories per mole to transition-state stabilization in enzymatic catalysis. The proposal invokes a large increase in hydrogen bond energy when the pKa values of the donor and acceptor (where Ka is the acid constant) become matched in the transition state (delta pKa=0). This hypothesis was tested by investigating the energetics of hydrogen bonds as a function of delta pKa for homologous series of compounds under nonaqueous conditions that are conducive to the formation of low-barrier hydrogen bonds. In all cases, there was a linear correlation between the increase in hydrogen-bond energy and the decrease in delta pKa, as expected from simple electrostatic effects. However, no additional energetic contribution to the hydrogen bond was observed at delta pKa=0. These results and those of other model studies suggest alternative mechanisms by which hydrogen bonds can contribute to enzymatic catalysis, in accord with conventional electrostatic considerations.

Catalysis↗
PubMed1996

pH dependencies of the Tetrahymena ribozyme reveal an unconventional origin of an apparent pKa.

The L-21 ScaI ribozyme derived from the Tetrahymena thermophila pre-rRNA group I intron catalyzes a site-specific endonucleolytic cleavage of RNA, DNA, and chimeric RNA/DNA oligonucleotides: CCCUCUA5 + G-->CCCUCU + GA5. The pH-rate dependence was determined for the reaction of the E.G complex with the oligonucleotide substrate d(CCCUC)r(U)d(A5) [(kcat/Km)S conditions]. Although it was shown that the pH dependence is not affected by specific buffers, there is inhibition by specific monovalent cations. The intrinsic pH-rate dependence is log-linear with slope 1 below pH 7, displays an apparent pKa of 7.6, remains nearly level until pH 8.5, and then begins to fall. Two models to explain the apparent pKa were ruled out: (1) the pKa represents loss of a proton from the nucleophilic 3' OH of G, and (2) the pKa arises from a change in rate-limiting step from a pH-dependent to a pH-independent step. In addition, these models, or others involving a single titration, cannot account for the decrease in activity at high pH. A third, unconventional, model is consistent with all of the data. It involves inactivation of the ribozyme by any of several independent titrations of groups with pKa values considerably higher than the apparent pKa of 7.6. The data are consistent with loss of catalytic function upon release of a proton from any one of 19 independent sites with pKa = 9.4 (the unperturbed pKa of N1 of G and N3 of U in solution). Independent experiments investigating the effect of pH on different reaction steps supported this model and suggested the identity of some of the required protons. This mechanism of inactivation is expected to generally affect the behavior of RNAs at pH values removed from the pKa of the titrating bases.

Animals↗
PubMed1996

Isolation of a local tertiary folding transition in the context of a globally folded RNA.

Binding of the Tetrahymena ribozyme's oligonucleotide substrate represents a local folding event in the context of a globally folded RNA. Substrate binding involves P1 duplex formation with the ribozyme's internal guide sequence to give an "open complex', followed by docking of the P1 duplex into tertiary interactions to give a "closed complex'. We have isolated the open complex as a thermodynamically stable species using a site-specific modification and high Na+ concentrations. This has allowed characterization of P1 docking, which represents a folding transition between local secondary and local tertiary structure. P1 docking is entropically driven, possibly accompanied by a release of bound water molecules. Strategies analogous to those described here can be used more generally to study local folding events in large structured RNAs and to explore the structural and energetic landscape for RNA folding.

Animals↗
PubMed1995

The nature of the transition state for enzyme-catalyzed phosphoryl transfer. Hydrolysis of O-aryl phosphorothioates by alkaline phosphatase.

There has been much speculation that enzymes change the nature of the transition state for phosphoryl transfer from the dissociative transition state observed in solution reactions to an associative transition state at the enzyme's active site. This proposal can be tested by comparing linear free energy relationships (LFERs) for nonenzymatic and enzymatic reactions, provided that the specificity of the enzyme's binding site does not perturb the dependence of rate on the intrinsic reactivity of a series of substrates. The shallow binding groove of Escherichia coli alkaline phosphatase (AP) and its wide specificity suggest that this enzyme may be suited for such an approach. A second requirement of this approach is that the actual chemical step is rate-limiting. Comparisons of the reactions of aryl phosphorothioates and aryl phosphates support the previous conclusion that a nonchemical step limits kcat/KM for reactions of aryl phosphates, but suggest that the chemical cleavage step is rate-limiting for the aryl phosphorothioates. We therefore determined the dependence of the rate of AP-catalyzed cleavage of a series of aryl phosphorothioates on the intrinsic reactivity of the substrates. The large negative values of beta leaving group = -0.8 for the enzymatic reaction (kcat/KM) and -1.1 for the nonenzymatic hydrolysis reaction suggest that there is considerable dissociative character in both the enzymatic and nonenzymatic transition states. Despite the wide specificity of AP, certain substrates deviate from the LFER, underscoring that extreme care is required in applying LFERs to enzymatic reactions. The large negative value of beta leaving group suggests that AP can achieve substantial catalysis via a transition state with dissociative character.

Alkaline Phosphatase↗
PubMed1995

Use of binding energy by an RNA enzyme for catalysis by positioning and substrate destabilization.

A fundamental catalytic principle for protein enzymes in the use of binding interactions away from the site of chemical transformation for catalysis. We have compared the binding and reactivity of a series of oligonucleotide substrates and products of the Tetrahymena ribozyme, which catalyzes a site-specific phosphodiester cleavage reaction: CCCUCUpA+G<-->CCCUCU-OH+GpA. The results suggest that this RNA enzyme, like protein enzymes, can utilize binding interactions to achieve substantial catalysis via entropic fixation and substrate destabilization. The stronger binding of the all-ribose oligonucleotide product compared to an analog with a terminal 3' deoxyribose residue gives an effective concentration of 2200 M for the 3' hydroxyl group, a value approaching those obtained with protein enzymes and suggesting the presence of a structurally well defined active site capable of precise positioning. The stabilization from tertiary binding interactions is 40-fold less for the oligonucleotide substrate than the oligonucleotide product, despite the presence of the reactive phosphoryl group in the substrate. This destabilization is accounted for by a model in which tertiary interactions away from the site of bond cleavage position the electron-deficient 3' bridging phosphoryl oxygen of the oligonucleotide substrate next to an electropositive Mg ion. As the phosphodiester bond breaks and this 3' oxygen atom develops a negative charge in the transition state, the weak interaction of the substrate with Mg2+ becomes strong. These strategies of "substrate destabilization" and "transition state stabilization" provide estimated rate enhancements of approximately 280- and approximately 60-fold, respectively. Analogous substrate destabilization by a metal ion or hydrogen bond donor may be used more generally by RNA and protein enzymes catalyzing reactions of phosphate esters.

Animals↗