Biomedical subjects

Daniel Herschlag

Publications and source records attributed to Daniel Herschlag.

35 records · Page 2Linked to original sources

PubMed2004

Do electrostatic interactions with positively charged active site groups tighten the transition state for enzymatic phosphoryl transfer?

The effect of electrostatic interactions on the transition-state character for enzymatic phosphoryl transfer has been a subject of much debate. In this work, we investigate the transition state for alkaline phosphatase (AP) using linear free-energy relationships (LFERs). We determined k(cat)/K(M) for a series of aryl sulfate ester monoanions to obtain the Brønsted coefficient, beta(lg), and compared the value to that obtained previously for a series of aryl phosphorothioate ester dianion substrates. Despite the difference in substrate charge, the observed Brønsted coefficients for AP-catalyzed aryl sulfate and aryl phosphorothioate hydrolysis (-0.76 +/- 0.14 and -0.77 +/- 0.10, respectively) are strikingly similar, with steric effects being responsible for the uncertainties in these values. Aryl sulfates and aryl phosphates react via similar loose transition states in solution. These observations suggest an apparent equivalency of the transition states for phosphorothioate and sulfate hydrolysis reactions at the AP active site and, thus, negligible effects of active site electrostatic interactions on charge distribution in the transition state.

Alkaline Phosphatase↗

PubMed2004

Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast.

Genes encoding RNA-binding proteins are diverse and abundant in eukaryotic genomes. Although some have been shown to have roles in post-transcriptional regulation of the expression of specific genes, few of these proteins have been studied systematically. We have used an affinity tag to isolate each of the five members of the Puf family of RNA-binding proteins in Saccharomyces cerevisiae and DNA microarrays to comprehensively identify the associated mRNAs. Distinct groups of 40-220 different mRNAs with striking common themes in the functions and subcellular localization of the proteins they encode are associated with each of the five Puf proteins: Puf3p binds nearly exclusively to cytoplasmic mRNAs that encode mitochondrial proteins; Puf1p and Puf2p interact preferentially with mRNAs encoding membrane-associated proteins; Puf4p preferentially binds mRNAs encoding nucleolar ribosomal RNA-processing factors; and Puf5p is associated with mRNAs encoding chromatin modifiers and components of the spindle pole body. We identified distinct sequence motifs in the 3'-untranslated regions of the mRNAs bound by Puf3p, Puf4p, and Puf5p. Three-hybrid assays confirmed the role of these motifs in specific RNA-protein interactions in vivo. The results suggest that combinatorial tagging of transcripts by specific RNA-binding proteins may be a general mechanism for coordinated control of the localization, translation, and decay of mRNAs and thus an integral part of the global gene expression program.

3' Untranslated Regions↗

PubMed2004

A base triple in the Tetrahymena group I core affects the reaction equilibrium via a threshold effect.

Previous work on group I introns has suggested that a central base triple might be more important for the first rather than the second step of self-splicing, leading to a model in which the base triple undergoes a conformational change during self-splicing. Here, we use the well-characterized L-21 ScaI ribozyme derived from the Tetrahymena group I intron to probe the effects of base-triple disruption on individual reaction steps. Consistent with previous results, reaction of a ternary complex mimicking the first chemical step in self-splicing is slowed by mutations in this base triple, whereas reaction of a ternary complex mimicking the second step of self-splicing is not. Paradoxically, mechanistic dissection of the base-triple disruption mutants indicates that active site binding is weakened uniformly for the 5'-splice site and the 5'-exon analog, mimics for the species bound in the first and second step of self-splicing. Nevertheless, the 5'-exon analog remains bound at the active site, whereas the 5'-splice site analog does not. This differential effect arises despite the uniform destabilization, because the wild-type ribozyme binds the 5'-exon analog more strongly in the active site than in the 5'-splice site analog. Thus, binding into the active site constitutes an additional barrier to reaction of the 5'-splice site analog, but not the 5'-exon analog, resulting in a reduced reaction rate constant for the first step analog, but not the second step analog. This threshold model explains the self-splicing observations without the need to invoke a conformational change involving the base triple, and underscores the importance of quantitative dissection for the interpretation of effects from mutations.

Animals↗

PubMed2003

The fastest global events in RNA folding: electrostatic relaxation and tertiary collapse of the Tetrahymena ribozyme.

Large RNAs can collapse into compact conformations well before the stable formation of the tertiary contacts that define their final folds. This study identifies likely physical mechanisms driving these early compaction events in RNA folding. We have employed time-resolved small-angle X-ray scattering to monitor the fastest global shape changes of the Tetrahymena ribozyme under different ionic conditions and with RNA mutations that remove long-range tertiary contacts. A partial collapse in each of the folding time-courses occurs within tens of milliseconds with either monovalent or divalent cations. Combined with comparison to predictions from structural models, this observation suggests a relaxation of the RNA to a more compact but denatured conformational ensemble in response to enhanced electrostatic screening at higher ionic concentrations. Further, the results provide evidence against counterion-correlation-mediated attraction between RNA double helices, a recently proposed model for early collapse. A previous study revealed a second 100 ms phase of collapse to a globular state. Surprisingly, we find that progression to this second early folding intermediate requires RNA sequence motifs that eventually mediate native long-range tertiary interactions, even though these regions of the RNA were observed to be solvent-accessible in previous footprinting studies under similar conditions. These results help delineate an analogy between the early conformational changes in RNA folding and the "burst phase" changes and molten globule formation in protein folding.

Animals↗

PubMed2003

Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA.

Docking of the P1 duplex into the pre-folded core of the Tetrahymena group I ribozyme exemplifies the formation of tertiary interactions in the context of a complex, structured RNA. We have applied Phi-analysis to P1 docking, which compares the effects of modifications on the rate constant for docking (k(dock)) with the effects on the docking equilibrium (K(dock)). To accomplish this we used a single molecule fluorescence resonance energy transfer assay that allows direct determination of the rate constants for formation of thermodynamically favorable, as well as unfavorable, states. Modification of the eight groups of the P1 duplex that make tertiary interactions with the core and changes in solution conditions decrease K(dock) up to 500-fold, whereas k(dock) changes by </=2-fold. The absence of effects on k(dock), both from atomic modifications and global perturbations, strongly suggests that the transition state for docking is early and does not closely resemble the docked state. These results, the slow rate of docking of 3s(-1), and the observation that a modification that is expected to increase the degrees of freedom between the P1 duplex and the ribozyme core accelerates docking, suggest a model in which a kinetic trap(s) slows docking substantially. Nonetheless, urea does not increase k(dock), suggesting that there is little change in the exposed surface area between the trapped, undocked state and the transition state. The findings highlight that urea and temperature dependencies can be inadequate to diagnose the presence of kinetic traps in a folding process. The results described here, combined with previous work, provide an in-depth view of an RNA tertiary structure formation event and suggest that large, highly structured RNAs may have local regions that are misordered.

Animals↗

PubMed2003

Challenges in enzyme mechanism and energetics.

Since the discovery of enzymes as biological catalysts, study of their enormous catalytic power and exquisite specificity has been central to biochemistry. Nevertheless, there is no universally accepted comprehensive description. Rather, numerous proposals have been presented over the past half century. The difficulty in developing a comprehensive description for the catalytic power of enzymes derives from the highly cooperative nature of their energetics, which renders impossible a simple division of mechanistic features and an absolute partitioning of catalytic contributions into independent and energetically additive components. Site-directed mutagenesis has emerged as an enormously powerful approach to probe enzymatic catalysis, illuminating many basic features of enzyme function and behavior. The emphasis of site-directed mutagenesis on the role of individual residues has also, inadvertently, limited experimental and conceptual attention to the fundamentally cooperative nature of enzyme function and energetics. The first part of this review highlights the structural and functional interconnectivity central to enzymatic catalysis. In the second part we ask: What are the features of enzymes that distinguish them from simple chemical catalysts? The answers are presented in conceptual models that, while simplified, help illustrate the vast amount known about how enzymes achieve catalysis. In the last section, we highlight the molecular and energetic questions that remain for future investigation and describe experimental approaches that will be necessary to answer these questions. The promise of advancing and integrating cutting edge conceptual, experimental, and computational tools brings mechanistic enzymology to a new era, one poised for novel fundamental insights into biological catalysis.

Binding Sites↗

PubMed2003

Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae.

We have analyzed the translational status of each mRNA in rapidly growing Saccharomyces cerevisiae. mRNAs were separated by velocity sedimentation on a sucrose gradient, and 14 fractions across the gradient were analyzed by quantitative microarray analysis, providing a profile of ribosome association with mRNAs for thousands of genes. For most genes, the majority of mRNA molecules were associated with ribosomes and presumably engaged in translation. This systematic approach enabled us to recognize genes with unusual behavior. For 43 genes, most mRNA molecules were not associated with ribosomes, suggesting that they may be translationally controlled. For 53 genes, including GCN4, CPA1, and ICY2, three genes for which translational control is known to play a key role in regulation, most mRNA molecules were associated with a single ribosome. The number of ribosomes associated with mRNAs increased with increasing length of the putative protein-coding sequence, consistent with longer transit times for ribosomes translating longer coding sequences. The density at which ribosomes were distributed on each mRNA (i.e., the number of ribosomes per unit ORF length) was well below the maximum packing density for nearly all mRNAs, consistent with initiation as the rate-limiting step in translation. Global analysis revealed an unexpected correlation: Ribosome density decreases with increasing ORF length. Models to account for this surprising observation are discussed.

Genome, Fungal↗

PubMed2003

Extraordinarily slow binding of guanosine to the Tetrahymena group I ribozyme: implications for RNA preorganization and function.

The Tetrahymena ribozyme derived from the self-splicing group I intron binds a 5'-splice site analog (S) and guanosine (G), catalyzing their conversion to a 5'-exon analog (P) and GA. Herein, we show that binding of guanosine is exceptionally slow, limiting the reaction at near neutral pH. Our results implicate a conformational rearrangement on guanosine binding, likely because the binding site is not prearranged in the absence of ligand. The fast accommodation of guanosine (10(2) to 10(3) x s(-1)) and prior structural data suggest local rather than global rearrangements, raising the possibility that folding of this and perhaps other large RNAs is not fully cooperative. Guanosine binding is accelerated by addition of residues that form helices, referred to as P9.0 and P10, immediately 5' and 3' to the guanosine. These rate enhancements provide evidence for binding intermediates that have the adjacent helices formed before accommodation of guanosine into its binding site. Because the ability to form the P9.0 and P10 helices distinguishes the guanosine at the correct 3'-splice site from other guanosine residues, the faster binding of the correct guanosine can enhance specificity of 3'-splice site selection. Thus, paradoxically, the absence of a preformed binding site and the resulting slow guanosine binding can contribute to splicing specificity by providing an opportunity for the adjacent helices to increase the rate of binding of the guanosine specifying the 3'-splice site.

Animals↗

PubMed2003

Adenosine 5'-O-(3-thio)triphosphate (ATPgammaS) is a substrate for the nucleotide hydrolysis and RNA unwinding activities of eukaryotic translation initiation factor eIF4A.

Whereas ATPgammaS is often considered a nonhydrolyzable substrate for ATPases, we present evidence that ATPgammaS is a good substrate for the RNA-stimulated nucleotide hydrolysis and RNA unwinding activities of eIF4A. In the presence of saturating single-stranded poly(U) RNA, eIF4A hydrolyzes ATPgammaS.Mg and ATP.Mg with similar steady-state parameters (KM(NTP.Mg) = 66 and 58 microM and kcat = 1.0 and 0.97 min(-1), respectively). ATPgammaS.Mg also supports catalysis of RNA unwinding within 10-fold of the rate supported by ATP.Mg. The identical steady-state rate parameters, in comparison with the expected difference in the intrinsic rate of hydrolysis for ATP and ATPgammaS, suggest a nonchemical rate-limiting step for nucleotide hydrolysis. These results raise caution concerning the assumption that ATPgammaS is a nonhydrolyzable ATP analog and underscore the utility of thio-substituted NTPs as mechanistic probes.

Adenosine Diphosphate↗

PubMed2002

Environmental effects on phosphoryl group bonding probed by vibrational spectroscopy: implications for understanding phosphoryl transfer and enzymatic catalysis.

We have used vibrational spectroscopy to study bonding in monosubstituted dianionic phosphates, both to learn more about basic properties intrinsic to this important class of biological substrates and to assess the ability of vibrational spectroscopy to provide a "sensor" or probe of the local environment experienced by the phosphoryl group. We examined the bonding properties of the phosphoryl group via vibrational spectroscopy for a series of compounds in which the phosphoryl substituent was varied systematically and extensively. A broad linear correlation of the bridging P-O(R) bond length and the pK(a) of the substituent alcohol was observed. The results indicate that the P-O(R) bond changes by only approximately 0.04 A with alcohol substituents that vary in pK(a) by approximately 12 units, suggesting that phosphoryl group bonding responds in a subtle but regular manner to changes in the local environment. We also determined the effect on the phosphoryl bonding from changes in the solvent environment. Addition of dimethyl sulfoxide (DMSO) elongates the bridging bond, presumably as a result of lessened solvation to the nonbridging oxygens and conservation of bond order to phosphorus. Finally, we have addressed the relationship between ground-state bonding properties and reactivity, as changing the leaving group substituent and adding DMSO have large rate effects, and it was previously proposed that lengthening of the bond to be broken is the cause of the increased reactivity. The results herein suggest, however, that the change in the bridging bond energy is small compared to the changes in energy that accompany the observed reactivity differences. Further analysis indicates that electrostatic interactions can provide a common driving force underlying both bond lengthening and the observed rate increases. We suggest that ground-state distortions of substrates bound to enzymes can provide a readout of the electrostatic active site environment, an environment that is otherwise difficult to assess.

Binding Sites↗

PubMed2002

Probing the Tetrahymena group I ribozyme reaction in both directions.

The Tetrahymena L-21 ScaI ribozyme derived from the self-splicing group I intron catalyzes a reversible reaction analogous to the first step of self-splicing: CCCUCUA (S) + [UC]G right harpoon over left harpoon CCCUCU (P) + [UC]GA. To relate our understanding of the ribozyme to the self-splicing reaction and to further the mechanistic dissection of the ribozyme reaction, we have established a quantitative kinetic and thermodynamic framework for the forward and reverse reaction of the L-21 ScaI ribozyme under identical conditions. Examination of the framework shows that binding of products is cooperative with binding enhanced 5-fold, as was observed previously for binding of the substrates. Further, binding of UCGA is 12-fold weaker than binding of the unphosphorylated UCG, analogous to the 20-fold weaker binding of phosphorylated S relative to P; the molecular interactions underlying the stronger binding of UCG were traced to the 3'-hydroxyl group of UCG. The symmetrical effects on binding of substrates and products result in the equilibrium between ribozyme-bound species, K(int), that is essentially unperturbed from the solution equilibrium, K(ext) (K(int) = [E.P.UCGA]/[E.S.UCG] = 4.6 and K(ext) = [P][UCGA]/[S][UCG] = 1.9). Last, we show that the pK(a) values of the nucleophiles in the forward and reverse reactions are >/=10. This observation suggests that metal ion activation of the nucleophile and stabilization of the leaving group can only account for a portion of the rate enhancement of this ribozyme. These and prior results suggest that the Tetrahymena group I ribozyme, analogous to protein enzymes, uses multiple catalytic strategies to achieve its large rate enhancement.

Animals↗

PubMed2002

Precision and functional specificity in mRNA decay.

Posttranscriptional processing of mRNA is an integral component of the gene expression program. By using DNA microarrays, we precisely measured the decay of each yeast mRNA, after thermal inactivation of a temperature-sensitive RNA polymerase II. The half-lives varied widely, ranging from approximately 3 min to more than 90 min. We found no simple correlation between mRNA half-lives and ORF size, codon bias, ribosome density, or abundance. However, the decay rates of mRNAs encoding groups of proteins that act together in stoichiometric complexes were generally closely matched, and other evidence pointed to a more general relationship between physiological function and mRNA turnover rates. The results provide strong evidence that precise control of the decay of each mRNA is a fundamental feature of the gene expression program in yeast.

Glycolysis↗

PubMed2002

RNA simulations: probing hairpin unfolding and the dynamics of a GNRA tetraloop.

Simulations of an RNA hairpin containing a GNRA tetraloop were conducted to allow the characterization of its secondary structure formation and dynamics. Ten 10 ns trajectories of the folded hairpin 5'-GGGC[GCAA]GCCU-3' were generated using stochastic dynamics and the GB/SA implicit solvent model at 300 K. Overall, we find the stem to be a very stable subunit of this molecule, whereas multiple loop conformations and transitions between them were observed. These trajectories strongly suggest that extension of the C6 base away from the loop occurs cooperatively with an N-type-->S-type sugar pucker conversion in that residue and that similar pucker transitions are necessary to stabilize other looped-out bases. In addition, a short-lived conformer with an extended fourth loop residue (A8) lacking this stabilizing 2'-endo pucker mode was observed. Results of thermal perturbation at 400 K support this model of loop dynamics. Unfolding trajectories were produced using this same methodology at temperatures of 500 to 700 K. The observed unfolding events display three-state behavior kinetically (including native, globular, and unfolded populations) and, based on these observations, we propose a folding mechanism that consists of three distinct events: (i) collapse of the random unfolded structure and sampling of the globular state; (ii) passage into the folded region of configurational space as stem base-pairs form and gain helicity; and (iii) attainment of proper loop geometry and organization of loop pairing and stacking interactions. These results are considered in the context of current experimental knowledge of this and similar nucleic acid hairpins.

Base Sequence↗

PubMed2002

Rapid compaction during RNA folding.

We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

Animals↗

PubMed2002

Alkaline phosphatase revisited: hydrolysis of alkyl phosphates.

Escherichia coli alkaline phosphatase (AP) is the prototypical two metal ion catalyst with two divalent zinc ions bound approximately 4 A apart in the active site. Studies spanning half a century have elucidated many structural and mechanistic features of this enzyme, rendering it an attractive model for investigating the potent catalytic power of bimetallic centers. Unfortunately, fundamental mechanistic features have been obscured by limitations with the standard assays. These assays generate concentrations of inorganic phosphate (P(i)) in excess of its inhibition constant (K(i) approximately 1 muM). This tight binding by P(i) has affected the majority of published kinetic constants. Furthermore, binding limits k(cat)/K(m) for reaction of p-nitrophenyl phosphate, the most commonly employed substrate. We describe a sensitive (32)P-based assay for hydrolysis of alkyl phosphates that avoids the complication of product inhibition. We have revisited basic mechanistic features of AP with these alkyl phosphate substrates. The results suggest that the chemical step for phosphorylation of the enzyme limits k(cat)/K(m). The pH-rate profile and additional results suggest that the serine nucleophile is active in its anionic form and has a pK(a) of < or = 5.5 in the free enzyme. An inactivating pK(a) of 8.0 is observed for binding of both substrates and inhibitors, and we suggest that this corresponds to ionization of a zinc-coordinated water molecule. Counter to previous suggestions, inorganic phosphate dianion appears to bind to the highly charged AP active site at least as strongly as the trianion. The dependence of k(cat)/K(m) on the pK(a) of the leaving group follows a Brønsted correlation with a slope of beta(lg) = -0.85 +/- 0.1, differing substantially from the previously reported value of -0.2 obtained from data with a less sensitive assay. This steep leaving group dependence is consistent with a largely dissociative transition state for AP-catalyzed hydrolysis of phosphate monoesters. The new (32)P-based assay employed herein will facilitate continued dissection of the AP reaction by providing a means to readily follow the chemical step for phosphorylation of the enzyme.

Alkaline Phosphatase↗

PubMed2002

Dissection of a metal-ion-mediated conformational change in Tetrahymena ribozyme catalysis.

Conformational changes are often required for the biological function of RNA molecules. In the Tetrahymena group I ribozyme reaction, a conformational change has been suggested to occur upon binding of the oligonucleotide substrate (S) or the guanosine nucleophile (G), leading to stronger binding of the second substrate. Recent work showed that the two substrates are bridged by a metal ion that coordinates both the nonbridging reactive phosphoryl oxygen of S and the 2'-OH of G. These results suggest that the energy from the metal ion substrate interactions is used to drive the proposed conformational change. In this work, we provide an experimental test for this model. The results provide strong support for the proposed conformational change and for a central role of the bridging metal ion in this change. The results from this work, combined with previous data, allow construction of a two-state model that quantitatively accounts for all of the observations in this and previous-work. This model provides a conceptual and quantitative framework that will facilitate understanding and further probing of the energetic and structural features of this conformational change and its role in catalysis.

Animals↗

PubMed2001

Exploring the folding landscape of a structured RNA.

Structured RNAs achieve their active states by traversing complex, multidimensional energetic landscapes. Here we probe the folding landscape of the Tetrahymena ribozyme by using a powerful approach: the folding of single ribozyme molecules is followed beginning from distinct regions of the folding landscape. The experiments, combined with small-angle x-ray scattering results, show that the landscape contains discrete folding pathways. These pathways are separated by large free-energy barriers that prevent interconversion between them, indicating that the pathways lie in deep channels in the folding landscape. Chemical protection and mutagenesis experiments are then used to elucidate the structural features that determine which folding pathway is followed. Strikingly, a specific long-range tertiary contact can either help folding or hinder folding, depending on when it is formed during the process. Together these results provide an unprecedented view of the topology of an RNA folding landscape and the RNA structural features that underlie this multidimensional landscape.

Animals↗