RESUMEN
Current methods to quantify the fraction of aminoacylated tRNAs, also known as the tRNA charge, are limited by issues with either low throughput, precision, and/or accuracy. Here, we present an optimized charge transfer RNA sequencing (tRNA-Seq) method that combines previous developments with newly described approaches to establish a protocol for precise and accurate tRNA charge measurements. We verify that this protocol provides robust quantification of tRNA aminoacylation and we provide an end-to-end method that scales to hundreds of samples including software for data processing. Additionally, we show that this method supports measurements of relative tRNA expression levels and can be used to infer tRNA modifications through reverse transcription misincorporations, thereby supporting multipurpose applications in tRNA biology.
Asunto(s)
ARN de Transferencia , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Aminoacilación de ARN de Transferencia , Análisis de Secuencia de ARN/métodos , Aminoacilación/genéticaRESUMEN
In the hypothetical RNA world, ribozymes could have acted as modern aminoacyl-tRNA synthetases (ARSs) to charge tRNAs, thus giving rise to the peptide synthesis along with the evolution of a primitive translation apparatus. We previously reported a T-boxzyme, Tx2.1, which selectively charges initiator tRNA with N-biotinyl-phenylalanine (BioPhe) in situ in a Flexible In-vitro Translation (FIT) system to produce BioPhe-initiating peptides. Here, we performed in vitro selection of elongation-capable T-boxzymes (elT-boxzymes), using para-azido-l-phenylalanine (PheAZ) as an acyl-donor. We implemented a new strategy to enrich elT-boxzyme-tRNA conjugates that self-aminoacylated on the 3'-terminus selectively. One of them, elT32, can charge PheAZ onto tRNA in trans in response to its cognate anticodon. Further evolution of elT32 resulted in elT49, with enhanced aminoacylation activity. We have demonstrated the translation of a PheAZ-containing peptide in an elT-boxzyme-integrated FIT system, revealing that elT-boxzymes are able to generate the PheAZ-tRNA in response to the cognate anticodon in situ of a custom-made translation system. This study, together with Tx2.1, illustrates a scenario where a series of ribozymes could have overseen aminoacylation and co-evolved with a primitive RNA-based translation system.
Asunto(s)
Anticodón , Biosíntesis de Proteínas , ARN Catalítico , Aminoacil-ARN de Transferencia , ARN Catalítico/metabolismo , ARN Catalítico/genética , Anticodón/genética , Aminoacil-ARN de Transferencia/metabolismo , Aminoacil-ARN de Transferencia/genética , Fenilalanina/metabolismo , Fenilalanina/análogos & derivados , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacil-ARNt Sintetasas/genética , Aminoacilación de ARN de Transferencia , Aminoacilación , Extensión de la Cadena Peptídica de TranslaciónRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that ligate amino acids to tRNAs, and often require editing to ensure accurate protein synthesis. Recessive mutations in aaRSs cause various neurological disorders in humans, yet the underlying mechanism remains poorly understood. Pathogenic aaRS mutations frequently cause protein destabilization and aminoacylation deficiency. In this study, we report that combined aminoacylation and editing defects cause severe proteotoxicity. We show that the ths1-C268A mutation in yeast threonyl-tRNA synthetase (ThrRS) abolishes editing and causes heat sensitivity. Surprisingly, experimental evolution of the mutant results in intragenic mutations that restore heat resistance but not editing. ths1-C268A destabilizes ThrRS and decreases overall Thr-tRNAThr synthesis, while the suppressor mutations in the evolved strains improve aminoacylation. We further show that deficiency in either ThrRS aminoacylation or editing is insufficient to cause heat sensitivity, and that ths1-C268A impairs ribosome-associated quality control. Our results suggest that aminoacylation deficiency predisposes cells to proteotoxic stress.
Asunto(s)
Aminoacil-ARNt Sintetasas , Estrés Proteotóxico , Humanos , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacilación , Mutación , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismo , Treonina-ARNt Ligasa/genéticaRESUMEN
The expanded hexanucleotide GGGGCC repeat mutation in the C9orf72 gene is the main genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Under one disease mechanism, sense and antisense transcripts of the repeat are predicted to bind various RNA-binding proteins, compromise their function and cause cytotoxicity. Here we identify phenylalanine-tRNA synthetase (FARS) subunit alpha (FARSA) as the main interactor of the CCCCGG antisense repeat RNA in cytosol. The aminoacylation of tRNAPhe by FARS is inhibited by antisense RNA, leading to decreased levels of charged tRNAPhe. Remarkably, this is associated with global reduction of phenylalanine incorporation in the proteome and decrease in expression of phenylalanine-rich proteins in cellular models and patient tissues. In conclusion, this study reveals functional inhibition of FARSA in the presence of antisense RNA repeats. Compromised aminoacylation of tRNA could lead to impairments in protein synthesis and further contribute to C9orf72 mutation-associated pathology.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Aminoacilación de ARN de Transferencia , Aminoacilación , Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genética , Proteína C9orf72/genética , Fenilalanina/genética , ARN de Transferencia de Fenilalanina , ARN sin SentidoRESUMEN
tRNA-bound amino acids often need to be identified, for instance, in cases where different amino acids compete for binding to the same tRNA. Here, we present a mass-spectrometry-based protocol to determine the amino acids bound to tRNA by aminoacylation. We detail how to perform the aminoacylation reaction, the preparation of the aminoacyl-tRNA for measurement, and the mass spectrometric analysis. We use arginine acylation as an example; however, this protocol can be applied to any other amino acid.
Asunto(s)
Aminoácidos , Aminoacil-ARNt Sintetasas , Aminoácidos/metabolismo , Aminoacilación , Aminoacil-ARNt Sintetasas/química , ARN de Transferencia/metabolismo , Espectrometría de MasasRESUMEN
The encoding step of translation involves attachment of amino acids to cognate tRNAs by aminoacyl-tRNA synthetases, themselves the product of coded peptide synthesis. So, the question arisesâbefore these enzymes evolved, how were primordial tRNAs selectively aminoacylated? Here, we demonstrate enzyme-free, sequence-dependent, chemoselective aminoacylation of RNA. We investigated two potentially prebiotic routes to aminoacyl-tRNA acceptor stem-overhang mimics and analyzed those oligonucleotides undergoing the most efficient aminoacylation. Overhang sequences do not significantly influence the chemoselectivity of aminoacylation by either route. For aminoacyl-transfer from a mixed anhydride donor strand, the chemoselectivity and stereoselectivity of aminoacylation depend on the terminal three base pairs of the stem. The results support early suggestions of a second genetic code in the acceptor stem.
Asunto(s)
Aminoacil-ARNt Sintetasas , ARN , ARN/metabolismo , Aminoacilación , Secuencia de Bases , Código Genético , ARN de Transferencia/química , Aminoacil-ARNt Sintetasas/metabolismo , Conformación de Ácido NucleicoRESUMEN
In Escherichia coli, inconsistencies between in vitro tRNA aminoacylation measurements and in vivo protein synthesis demands were postulated almost 40 years ago, but have proven difficult to confirm. Whole-cell modeling can test whether a cell behaves in a physiologically correct manner when parameterized with in vitro measurements by providing a holistic representation of cellular processes in vivo. Here, a mechanistic model of tRNA aminoacylation, codon-based polypeptide elongation, and N-terminal methionine cleavage was incorporated into a developing whole-cell model of E. coli. Subsequent analysis confirmed the insufficiency of aminoacyl-tRNA synthetase kinetic measurements for cellular proteome maintenance, and estimated aminoacyl-tRNA synthetase kcats that were on average 7.6-fold higher. Simulating cell growth with perturbed kcats demonstrated the global impact of these in vitro measurements on cellular phenotypes. For example, an insufficient kcat for HisRS caused protein synthesis to be less robust to the natural variability in aminoacyl-tRNA synthetase expression in single cells. More surprisingly, insufficient ArgRS activity led to catastrophic impacts on arginine biosynthesis due to underexpressed N-acetylglutamate synthase, where translation depends on repeated CGG codons. Overall, the expanded E. coli model deepens understanding of how translation operates in an in vivo context.
Asunto(s)
Aminoacil-ARNt Sintetasas , Arginina , Escherichia coli , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacilación , Arginina/biosíntesis , Escherichia coli/metabolismo , Retroalimentación , Aminoacilación de ARN de TransferenciaRESUMEN
This chapter describes the preparation of pre-charged Arg-tRNA that can be used in arginylation reaction. While in a typical arginylation reaction arginyl-tRNA synthetase (RARS) is normally included as a component of the reaction and continually charges tRNA during arginylation, it is sometimes necessary to separate the charging and the arginylation step, in order to perform each reaction under controlled conditions, e.g., for measuring the kinetics or determining the effect of different compounds and chemicals on the reaction. In such cases, tRNAArg can be pre-charged with Arg and purified away from the RARS enzyme prior to arginylation.
Asunto(s)
Aminoacil-ARNt Sintetasas , Arginino-ARNt Ligasa , Arginino-ARNt Ligasa/química , Arginino-ARNt Ligasa/genética , Arginino-ARNt Ligasa/metabolismo , ARN de Transferencia de Arginina/química , ARN de Transferencia de Arginina/genética , ARN de Transferencia de Arginina/metabolismo , Aminoacilación , ARN de Transferencia/genética , Aminoacilación de ARN de Transferencia , Cinética , Aminoacil-ARNt Sintetasas/metabolismoRESUMEN
Aspartyl-tRNA synthetase catalyzes the attachment of aspartic acid to its cognate tRNA by the aminoacylation reaction during the initiation of the protein biosynthesis process. In the second step of the aminoacylation reaction, known as the charging step, the aspartate moiety is transferred from aspartyl-adenylate to the 3'-OH of A76 of tRNA through a proton transfer process. We have investigated different pathways for the charging step through three separate QM/MM simulations combined with the enhanced sampling method of well-sliced metadynamics and found out the most feasible pathway for the reaction at the active site of the enzyme. In the charging reaction, both the phosphate group and the ammonium group after deprotonation can potentially act as a base for proton transfer in the substrate-assisted mechanism. We have considered three possible mechanisms involving different pathways of proton transfer, and only one of them is determined to be enzymatically feasible. The free energy landscape along reaction coordinates where the phosphate group acts as the general base showed that, in the absence of water, the barrier height is 52.6 kcal/mol. The free energy barrier is reduced to 39.7 kcal/mol when the active site water molecules are also treated quantum mechanically, thus allowing a water mediated proton transfer. The charging reaction involving the ammonium group of the aspartyl adenylate is found to follow a path where first a proton from the ammonium group moves to a water in the vicinity forming a hydronium ion (H3O+) and NH2 group. The hydronium ion subsequently passes the proton to the Asp233 residue, thus minimizing the chance of back proton transfer from hydronium to the NH2 group. The neutral NH2 group subsequently takes the proton from the O3' of A76 with a free energy barrier of 10.7 kcal/mol. In the next step, the deprotonated O3' makes a nucleophilic attack to the carbonyl carbon forming a tetrahedral transition state with a free energy barrier of 24.8 kcal/mol. Thus, the present work shows that the charging step proceeds through a multiple proton transfer mechanism where the amino group formed after deprotonation acts as the base to capture a proton from O3' of A76 rather than the phosphate group. The current study also shows the important role played by Asp233 in the proton transfer process.
Asunto(s)
Aspartato-ARNt Ligasa , Dominio Catalítico , Protones , Aminoacilación , Agua/química , ARN de Transferencia/química , FosfatosRESUMEN
tRNAs are key partners in ribosome-dependent protein synthesis. This process is highly dependent on the fidelity of tRNA aminoacylation by aminoacyl-tRNA synthetases and relies primarily on sets of identities within tRNA molecules composed of determinants and antideterminants preventing mischarging by non-cognate synthetases. Such identity sets were discovered in the tRNAs of a few model organisms, and their properties were generalized as universal identity rules. Since then, the panel of identity elements governing the accuracy of tRNA aminoacylation has expanded considerably, but the increasing number of reported functional idiosyncrasies has led to some confusion. In parallel, the description of other processes involving tRNAs, often well beyond aminoacylation, has progressed considerably, greatly expanding their interactome and uncovering multiple novel identities on the same tRNA molecule. This review highlights key findings on the mechanistics and evolution of tRNA and tRNA-like identities. In addition, new methods and their results for searching sets of multiple identities on a single tRNA are discussed. Taken together, this knowledge shows that a comprehensive understanding of the functional role of individual and collective nucleotide identity sets in tRNA molecules is needed for medical, biotechnological and other applications.
Asunto(s)
Aminoacil-ARNt Sintetasas , Aminoacilación de ARN de Transferencia , Aminoacilación , Biotecnología , ARN de TransferenciaRESUMEN
Histidyl-tRNA synthetase (HARS) ligates histidine to its cognate transfer RNA (tRNAHis). Mutations in HARS cause the human genetic disorders Usher syndrome type 3B (USH3B) and Charcot-Marie-Tooth syndrome type 2W (CMT2W). Treatment for these diseases remains symptomatic, and no disease specific treatments are currently available. Mutations in HARS can lead to destabilization of the enzyme, reduced aminoacylation, and decreased histidine incorporation into the proteome. Other mutations lead to a toxic gain-of-function and mistranslation of non-cognate amino acids in response to histidine codons, which can be rescued by histidine supplementation in vitro. We discuss recent advances in characterizing HARS mutations and potential applications of amino acid and tRNA therapy for future gene and allele specific therapy.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Histidina , Humanos , Histidina/genética , Histidina/metabolismo , Mutación , Histidina-ARNt Ligasa/genética , Enfermedad de Charcot-Marie-Tooth/genética , AminoacilaciónRESUMEN
Aminoacyl-tRNA synthetases are enzymes that ensure accurate protein synthesis. Variants of the dual-functional cytoplasmic human glutamyl-prolyl-tRNA synthetase, EPRS1, have been associated with leukodystrophy, diabetes and bone disease. Here, we report compound heterozygous variants in EPRS1 in a 4-year-old female patient presenting with psychomotor developmental delay, seizures and deafness. Functional studies of these two missense mutations support major defects in enzymatic function in vitro and contributed to confirmation of the diagnosis.
Asunto(s)
Aminoacil-ARNt Sintetasas , Sordera , Epilepsia , Femenino , Humanos , Preescolar , Aminoacilación , Aminoacil-ARNt Sintetasas/genética , Mutación , Epilepsia/diagnóstico , Epilepsia/genética , Convulsiones/genética , Sordera/genéticaRESUMEN
Transfer RNA (tRNA) utilizes multiple properties of abundance, modification, and aminoacylation in translational regulation. These properties were typically studied one-by-one; however, recent advance in high throughput tRNA sequencing enables their simultaneous assessment in the same sequencing data. How these properties are coordinated at the transcriptome level is an open question. Here, we develop a single-read tRNA analysis pipeline that takes advantage of the pseudo single-molecule nature of tRNA sequencing in NGS libraries. tRNAs are short enough that a single NGS read can represent one tRNA molecule, and can simultaneously report on the status of multiple modifications, aminoacylation, and fragmentation of each molecule. We find correlations among modification-modification, modification-aminoacylation and modification-fragmentation. We identify interdependencies among one of the most common tRNA modifications, m1A58, as coordinators of tissue-specific gene expression. Our method, SingLe-read Analysis of Crosstalks (SLAC), reveals tRNAome-wide networks of modifications, aminoacylation, and fragmentation. We observe changes of these networks under different stresses, and assign a function for tRNA modification in translational regulation and fragment biogenesis. SLAC leverages the richness of the tRNA-seq data and provides new insights on the coordination of tRNA properties.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , ARN de Transferencia , Aminoacilación , ARN de Transferencia/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
tRNA is a key component in life's most fundamental process, the translation of the instructions contained in mRNA into proteins. Its role had to be executed as soon as the earliest translation emerged, but the questions of the prebiotic tRNA materialization, aminoacylation, and the origin of the coding triplets it carries are still open. Here, these questions are addressed by utilizing a distinct pattern of coding triplets highly conserved in the acceptor stems from the modern bacterial tRNAs of five early-emerging amino acids. Self-assembly of several copies of a short RNA oligonucleotide that carries a related pattern of coding triplets, via a simple and statistically feasible process, is suggested to result in a proto-tRNA model highly compatible with the cloverleaf secondary structure of the modern tRNA. Furthermore, these stem coding triplets evoke the possibility that they were involved in self-aminoacylation of proto-tRNAs prior to the emergence of the earliest synthetases, a process proposed to underlie the formation of the genetic code. Being capable of autonomous materialization and of self-aminoacylation, this verifiable model of the proto-tRNA advent adds principal components to an initial set of molecules and processes that may have led, exclusively through natural means, to the emergence of life.
Asunto(s)
Aminoacil-ARNt Sintetasas , ARN de Transferencia , Aminoacilación , ARN de Transferencia/metabolismo , Código Genético , ARN/metabolismo , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/genética , Conformación de Ácido Nucleico , Evolución MolecularRESUMEN
Mitochondrial translation is of high significance for cellular energy homeostasis. Aminoacyl-tRNA synthetases (aaRSs) are crucial translational components. Mitochondrial aaRS variants cause various human diseases. However, the pathogenesis of the vast majority of these diseases remains unknown. Here, we identified two novel SARS2 (encoding mitochondrial seryl-tRNA synthetase) variants that cause a multisystem disorder. c.654-14T > A mutation induced mRNA mis-splicing, generating a peptide insertion in the active site; c.1519dupC swapped a critical tRNA-binding motif in the C-terminus due to stop codon readthrough. Both mutants exhibited severely diminished tRNA binding and aminoacylation capacities. A marked reduction in mitochondrial tRNASer(AGY) was observed due to RNA degradation in patient-derived induced pluripotent stem cells (iPSCs), causing impaired translation and comprehensive mitochondrial function deficiencies. These impairments were efficiently rescued by wild-type SARS2 overexpression. Either mutation caused early embryonic fatality in mice. Heterozygous mice displayed reduced muscle tissue-specific levels of tRNASers. Our findings elucidated the biochemical and cellular consequences of impaired translation mediated by SARS2, suggesting that reduced abundance of tRNASer(AGY) is a key determinant for development of SARS2-related diseases.
Asunto(s)
Aminoacil-ARNt Sintetasas , COVID-19 , Serina-ARNt Ligasa , Humanos , Ratones , Animales , ARN de Transferencia de Serina/genética , Serina-ARNt Ligasa/genética , Serina-ARNt Ligasa/metabolismo , Aminoacil-ARNt Sintetasas/genética , AminoacilaciónRESUMEN
There have been significant advancements in radical-mediated reactions through covalent-based organocatalysis. Here, we present the generation of iminyl and amidyl radicals via N-heterocyclic carbene (NHC) catalysis, enabling diastereoselective aminoacylation of trisubstituted alkenes. Different from photoredox catalysis, single electron transfer from the deprotonated Breslow intermediate to O-aryl hydroxylamine generates an NHC-bound ketyl radical, which undergoes diastereocontrolled cross-coupling with the prochiral C-centered radical. This operationally simple method provides a straightforward access to a variety of pyrroline and oxazolidinone heterocycles with vicinal stereocenters (77 examples, up to >19:1 d.r.). Electrochemical studies of the acyl thiazolium salts support our reaction design and highlight the reducing ability of Breslow-type derivatives. A detailed computational analysis of this organocatalytic system suggests that radical-radical coupling is the rate-determining step, in which π-π stacking interaction between the radical intermediates subtly controls the diastereoselectivity.
Asunto(s)
Alquenos , Aminoacilación , CatálisisRESUMEN
TriTryp diseases (Leishmaniasis, Human African Trypanosomiasis (HAT), and Chagas disease) are devastating parasitic neglected tropical diseases (NTDs) that affect billions of people in developing countries, cause high mortality in humans, and impose a large socio-economic burden. The current treatment options against tritryp diseases are suboptimal and challenging due to the emergence of resistance against available tritryp drugs. Hence, designing and developing effective anti-tritryp drugs with novel targets are required. Aminoacyl-tRNA synthetases (AARSs) involved in specific aminoacylation of transfer RNAs (tRNAs), interrupt protein synthesis through inhibitors, and retard the parasite growth. AaRSs have long been studied as therapeutic targets in bacteria, and three aaRS inhibitors, mupirocin (against IleRS), tavaborole AN2690 (against LeuRS), and halofuginone (against ProRS), are already in clinical practice. The structural differences between tritryp and human aaRSs and the presence of unique sequences (N-terminal domain/C-terminal domain/catalytic domain) make them potential target for developing selective inhibitors. Drugs based on a single aaRS target developed by high-throughput screening (HTS) are less effective due to the emergence of resistance. However, designing multi-targeted drugs may be a better strategy for resistance development. In this perspective, we discuss the characteristics of tritryp aaRSs, sequence conservation in their orthologs and their peculiarities, recent advancements towards the single-target and multi-target aaRS inhibitors developed through rational design.
Asunto(s)
Aminoacil-ARNt Sintetasas , Enfermedad de Chagas , Leishmaniasis , Tripanosomiasis Africana , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacilación , Animales , Humanos , Leishmaniasis/tratamiento farmacológico , Enfermedades Desatendidas/tratamiento farmacológico , Tripanosomiasis Africana/tratamiento farmacológicoRESUMEN
Human mitochondrial gene expression relies on the specific recognition and aminoacylation of mitochondrial tRNAs (mtRNAs) by nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs). Despite their essential role in cellular energy homeostasis, strong mutation pressure and genetic drift have led to an unparalleled sequence erosion of animal mtRNAs. The structural and functional consequences of this erosion are not understood. Here, we present cryo-EM structures of the human mitochondrial seryl-tRNA synthetase (mSerRS) in complex with mtRNASer(GCU). These structures reveal a unique mechanism of substrate recognition and aminoacylation. The mtRNASer(GCU) is highly degenerated, having lost the entire D-arm, tertiary core, and stable L-shaped fold that define canonical tRNAs. Instead, mtRNASer(GCU) evolved unique structural innovations, including a radically altered T-arm topology that serves as critical identity determinant in an unusual shape-selective readout mechanism by mSerRS. Our results provide a molecular framework to understand the principles of mito-nuclear co-evolution and specialized mechanisms of tRNA recognition in mammalian mitochondrial gene expression.
Asunto(s)
Aminoacil-ARNt Sintetasas , ARN de Transferencia , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacilación/genética , Animales , Humanos , Mamíferos/genética , Mitocondrias/metabolismo , ARN Mitocondrial/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
The process of protein biosynthesis is initiated by the aminoacylation process where a transfer ribonucleic acid (tRNA) is charged by the attachment of its cognate amino acid at the active site of the corresponding aminoacyl tRNA synthetase enzyme. The first step of the aminoacylation process, known as the adenylation reaction, involves activation of the cognate amino acid where it reacts with a molecule of adenosine triphosphate (ATP) at the active site of the enzyme to form the aminoacyl adenylate and inorganic pyrophosphate. In the current work, we have investigated the adenylation reaction between aspartic acid and ATP at the active site of the fully solvated aspartyl tRNA synthetase (AspRS) from Escherichia coli in aqueous medium at room temperature through hybrid quantum mechanical/molecular mechanical (QM/MM) simulations combined with enhanced sampling methods of well-tempered and well-sliced metadynamics. The objective of the present work is to study the associated free energy landscape and reaction barrier and also to explore the effects of active site mutation on the free energy surface of the reaction. The current calculations include finite temperature effects on free energy profiles. In particular, apart from contributions of interaction energies, the current calculations also include contributions of conformational, vibrational, and translational entropy of active site residues, substrates, and also the rest of the solvated protein and surrounding water into the free energy calculations. The present QM/MM metadynamics simulations predict a free energy barrier of 23.35 and 23.5 kcal/mol for two different metadynamics methods used to perform the reaction at the active site of the wild type enzyme. The free energy barrier increases to 30.6 kcal/mol when Arg217, which is an important conserved residue of the wild type enzyme at its active site, is mutated by alanine. These free energy results including the effect of mutation compare reasonably well with those of kinetic experiments that are available in the literature. The current work also provides molecular details of structural changes of the reactants and surroundings as the system dynamically evolves along the reaction pathway from reactant to the product state through QM/MM metadynamics simulations.
Asunto(s)
Aspartato-ARNt Ligasa , Adenosina Trifosfato/metabolismo , Aminoácidos/metabolismo , Aminoacilación , Aspartato-ARNt Ligasa/química , Aspartato-ARNt Ligasa/genética , Aspartato-ARNt Ligasa/metabolismo , Sitios de Unión , Dominio Catalítico , Entropía , Escherichia coli/genética , Ligasas/metabolismo , ARN de Transferencia/metabolismoRESUMEN
Systems of catalytic RNAs presumably gave rise to important evolutionary innovations, such as the genetic code. Such systems may exhibit particular tolerance to errors (error minimization) as well as coding specificity. While often assumed to result from natural selection, error minimization may instead be an emergent by-product. In an RNA world, a system of self-aminoacylating ribozymes could enforce the mapping of amino acids to anticodons. We measured the activity of thousands of ribozyme mutants on alternative substrates (activated analogs for tryptophan, phenylalanine, leucine, isoleucine, valine, and methionine). Related ribozymes exhibited shared preferences for substrates, indicating that adoption of additional amino acids by existing ribozymes would itself lead to error minimization. Furthermore, ribozyme activity was positively correlated with specificity, indicating that selection for increased activity would also lead to increased specificity. These results demonstrate that by-products of ribozyme evolution could lead to adaptive value in specificity and error tolerance.