Evolution Of aminoacyl-tRNA Synthetases Research Articles

Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation.

Genetic code expansion (GCE) has become a critical tool in biology by enabling the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons ( e.g ., the amber stop codon). Many orthogonal aaRS/tRNA pairs covering a wide range of ncAAs have been generated by directed evolution, yet the evolution of new aaRS/tRNA pairs by standard strategies remains a labor-intensive process that often produces aaRS/tRNA pairs with suboptimal ncAA incorporation efficiencies. In this study, we present a strategy for evolving aaRSs that leverages OrthoRep to drive their continuous hypermutation in yeast. We demonstrate our strategy in 8 independent aaRS evolution campaigns starting from 4 different aaRS/tRNA parents targeting 7 distinct ncAAs. We observed the rapid evolution of multiple novel aaRSs capable of incorporating an overall range of 13 ncAAs tested into proteins in response to the amber codon. Some evolved systems reached efficiencies for amber codon-specified ncAA-dependent translation comparable to translation with natural amino acids specified by sense codons in yeast. Additionally, we discovered a surprising aaRS that evolved to self-regulate its own expression for greater dependency on ncAAs for translation. These findings demonstrate the potential of OrthoRep-driven aaRS evolution platforms in supporting the continued growth of GCE technologies.

Aminoacyl-tRNA Synthetase Evolution within the Dynamic Tripartite Translation System of Plant Cells.

Eukaryotes maintain separate protein translation systems for nuclear and organellar genes, including distinct sets of tRNAs and aminoacyl-tRNA synthetases (aaRSs). In animals, mitochondrial-targeted aaRSs are expressed at lower levels and are less conserved in sequence than cytosolic aaRSs involved in translation of nuclear mRNAs, likely reflecting lower translational demands in mitochondria. In plants, translation is further complicated by the presence of plastids, which share most aaRSs with mitochondria. In addition, plant mitochondrial tRNA pools have a dynamic history of gene loss and functional replacement by tRNAs from other compartments. To investigate the consequences of these distinctive features of translation in plants, we analyzed sequence evolution in angiosperm aaRSs. In contrast to previously studied eukaryotic systems, we found that plant organellar and cytosolic aaRSs exhibit only a small difference in expression levels, and organellar aaRSs are slightly more conserved than cytosolic aaRSs. We hypothesize that these patterns result from high translational demands associated with photosynthesis in mature chloroplasts. We also investigated aaRS evolution in Sileneae, an angiosperm lineage with extensive mitochondrial tRNA replacement and aaRS retargeting. We predicted positive selection for changes in aaRS sequence resulting from these recent changes in subcellular localization and tRNA substrates but found little evidence for accelerated sequence divergence. Overall, the complex tripartite translation system in plant cells appears to have imposed more constraints on the long-term evolutionary rates of organellar aaRSs compared with other eukaryotic lineages, and plant aaRS protein sequences appear largely robust to more recent perturbations in subcellular localization and tRNA interactions.

Did Amino Acid Side Chain Reactivity Dictate the Composition and Timing of Aminoacyl-tRNA Synthetase Evolution?

The twenty amino acids in the standard genetic code were fixed prior to the last universal common ancestor (LUCA). Factors that guided this selection included establishment of pathways for their metabolic synthesis and the concomitant fixation of substrate specificities in the emerging aminoacyl-tRNA synthetases (aaRSs). In this conceptual paper, we propose that the chemical reactivity of some amino acid side chains (e.g., lysine, cysteine, homocysteine, ornithine, homoserine, and selenocysteine) delayed or prohibited the emergence of the corresponding aaRSs and helped define the amino acids in the standard genetic code. We also consider the possibility that amino acid chemistry delayed the emergence of the glutaminyl- and asparaginyl-tRNA synthetases, neither of which are ubiquitous in extant organisms. We argue that fundamental chemical principles played critical roles in fixation of some aspects of the genetic code pre- and post-LUCA.

G:U-Independent RNA Minihelix Aminoacylation by Nanoarchaeum equitans Alanyl-tRNA Synthetase: An Insight into the Evolution of Aminoacyl-tRNA Synthetases

Nanoarchaeum equitans is a species of hyperthermophilic archaea with the smallest genome size. Its alanyl-tRNA synthetase genes are split into AlaRS-α and AlaRS-β, encoding the respective subunits. In the current report, we surveyed N. equitans AlaRS-dependent alanylation of RNA minihelices, composed only of the acceptor stem and the T-arm of tRNAAla. Combination of AlaRS-α and AlaRS-β showed a strong alanylation activity specific to a single G3:U70 base pair, known to mark a specific tRNA for charging with alanine. However, AlaRS-α alone had a weak but appreciable alanylation activity that was independent of the G3:U70 base pair. The shorter 16-mer RNA tetraloop substrate mimicking only the first four base pairs of the acceptor stem of tRNAAla, but with C3:G70 base pair, was also successfully aminoacylated by AlaRS-α. The end of the acceptor stem, including CCA-3ʹ terminus and the discriminator A73, was able to function as a minimal structure for the recognition by the enzyme. Our findings imply that aminoacylation by N. equitans AlaRS-α may represent a vestige of a primitive aminoacylation system, before the appearance of the G3:U70 pair as an identity element for alanine.

Evolution of Life on Earth: tRNA, Aminoacyl-tRNA Synthetases and the Genetic Code.

Life on Earth and the genetic code evolved around tRNA and the tRNA anticodon. We posit that the genetic code initially evolved to synthesize polyglycine as a cross-linking agent to stabilize protocells. We posit that the initial amino acids to enter the code occupied larger sectors of the code that were then invaded by incoming amino acids. Displacements of amino acids follow selection rules. The code sectored from a glycine code to a four amino acid code to an eight amino acid code to an ~16 amino acid code to the standard 20 amino acid code with stops. The proposed patterns of code sectoring are now most apparent from patterns of aminoacyl-tRNA synthetase evolution. The Elongation Factor-Tu GTPase anticodon-codon latch that checks the accuracy of translation appears to have evolved at about the eight amino acid to ~16 amino acid stage. Before evolution of the EF-Tu latch, we posit that both the 1st and 3rd anticodon positions were wobble positions. The genetic code evolved via tRNA charging errors and via enzymatic modifications of amino acids joined to tRNAs, followed by tRNA and aminoacyl-tRNA synthetase differentiation. Fidelity mechanisms froze the code by inhibiting further innovation.

The evolution of aminoacyl-tRNA synthetases: From dawn to LUCA.

The origin of all extant life on earth is intimately linked to the establishment of the principal components of the Genetic Code. Aminoacyl-tRNA synthetases (aaRS), by virtue of their universality and essential functions in protein synthesis, count among the biomolecules that evolved to a level of complexity comparable to their extant state before the advent of the Last Universal Common Ancestor (LUCA). Despite the enormous technical difficulties in analyzing such an ancient process, proposals have been put forward to describe the emergence and evolution of the two aaRS families. In this chapter, I critically review some of these proposals and place them along a hypothetical timeline based on other essential aspects of the origin of life. This chapter focuses on the evolution of the aaRS prior to LUCA. Readers will be referred to excellent literature that covers the phylogeny of aaRS in the three extant domains of life.

Plasticity and Constraints of tRNA Aminoacylation Define Directed Evolution of Aminoacyl-tRNA Synthetases.

Genetic incorporation of noncanonical amino acids (ncAAs) has become a powerful tool to enhance existing functions or introduce new ones into proteins through expanded chemistry. This technology relies on the process of nonsense suppression, which is made possible by directing aminoacyl-tRNA synthetases (aaRSs) to attach an ncAA onto a cognate suppressor tRNA. However, different mechanisms govern aaRS specificity toward its natural amino acid (AA) substrate and hinder the engineering of aaRSs for applications beyond the incorporation of a single l-α-AA. Directed evolution of aaRSs therefore faces two interlinked challenges: the removal of the affinity for cognate AA and improvement of ncAA acylation. Here we review aspects of AA recognition that directly influence the feasibility and success of aaRS engineering toward d- and β-AAs incorporation into proteins in vivo. Emerging directed evolution methods are described and evaluated on the basis of aaRS active site plasticity and its inherent constraints.

A tRNA- and Anticodon-Centric View of the Evolution of Aminoacyl-tRNA Synthetases, tRNAomes, and the Genetic Code.

Pathways of standard genetic code evolution remain conserved and apparent, particularly upon analysis of aminoacyl-tRNA synthetase (aaRS) lineages. Despite having incompatible active site folds, class I and class II aaRS are homologs by sequence. Specifically, structural class IA aaRS enzymes derive from class IIA aaRS enzymes by in-frame extension of the protein N-terminus and by an alternate fold nucleated by the N-terminal extension. The divergence of aaRS enzymes in the class I and class II clades was analyzed using the Phyre2 protein fold recognition server. The class I aaRS radiated from the class IA enzymes, and the class II aaRS radiated from the class IIA enzymes. The radiations of aaRS enzymes bolster the coevolution theory for evolution of the amino acids, tRNAomes, the genetic code, and aaRS enzymes and support a tRNA anticodon-centric perspective. We posit that second- and third-position tRNA anticodon sequence preference (C>(U~G)>A) powerfully selected the sectoring pathway for the code. GlyRS-IIA appears to have been the primordial aaRS from which all aaRS enzymes evolved, and glycine appears to have been the primordial amino acid around which the genetic code evolved.

In Silico Evolution of Aminoacyl-tRNA Synthetases for Incorporation of Noncanonical Amino Acids

In Silico Evolution of Aminoacyl-tRNA Synthetases for Incorporation of Noncanonical Amino Acids

Aminoacyl-tRNA synthetase evolution and sectoring of the genetic code

ABSTRACTThe genetic code sectored via tRNA charging errors, and the code progressed toward closure and universality because of evolution of aminoacyl-tRNA synthetase (aaRS) fidelity and translational fidelity mechanisms. Class I and class II aaRS folds are identified as homologs. From sequence alignments, a structurally conserved Zn-binding domain common to class I and class II aaRS was identified. A model for the class I and class II aaRS alternate folding pathways is posited. Five mechanisms toward code closure are highlighted: 1) aaRS proofreading to remove mischarged amino acids from tRNA; 2) accurate aaRS active site specification of amino acid substrates; 3) aaRS-tRNA anticodon recognition; 4) conformational coupling proofreading of the anticodon-codon interaction; and 5) deamination of tRNA wobble adenine to inosine. In tRNA anticodons there is strong wobble sequence preference that results in a broader spectrum of contacts to synonymous mRNA codon wobble bases. Adenine is excluded from the anticodon wobble position of tRNA unless it is modified to inosine. Uracil is generally preferred to cytosine in the tRNA anticodon wobble position. Because of wobble ambiguity when tRNA reads mRNA, the maximal coding capacity of the three nucleotide code read by tRNA is 31 amino acids + stops.

Abiogenic evolution of aminoacyl-tRNA synthetase

Abiogenic evolution of aminoacyl-tRNA synthetase

Continuous directed evolution of aminoacyl-tRNA synthetases.

Directed evolution of orthogonal aminoacyl-tRNA synthetases (AARSs) enables site-specific installation of non-canonical amino acids (ncAAs) into proteins. Traditional evolution techniques typically produce AARSs with greatly reduced activity and selectivity compared to their wild-type counterparts. We designed phage-assisted continuous evolution (PACE) selections to rapidly produce highly active and selective orthogonal AARSs through hundreds of generations of evolution. PACE of a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) improved its enzymatic efficiency (kcat/KMtRNA) 45-fold compared to the parent enzyme. Transplantation of the evolved mutations into other PylRS-derived synthetases improved yields of proteins containing non-canonical residues up to 9.7-fold. Simultaneous positive and negative selection PACE over 48 h greatly improved the selectivity of a promiscuous Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant for site-specific incorporation of p-iodo-L-phenylalanine. These findings offer new AARSs that increase the utility of orthogonal translation systems and establish the capability of PACE to efficiently evolve orthogonal AARSs with high activity and amino acid specificity.

The Roles of Compensatory Evolution and Constraint in Aminoacyl tRNA Synthetase Evolution.

Mitochondrial protein translation requires interactions between transfer RNAs encoded by the mitochondrial genome (mt-tRNAs) and mitochondrial aminoacyl tRNA synthetase proteins (mt-aaRS) encoded by the nuclear genome. It has been argued that animal mt-tRNAs have higher deleterious substitution rates relative to their nuclear-encoded counterparts, the cytoplasmic tRNAs (cyt-tRNAs). This dynamic predicts elevated rates of compensatory evolution of mt-aaRS that interact with mt-tRNAs, relative to aaRS that interact with cyt-tRNAs (cyt-aaRS). We find that mt-aaRS do evolve at significantly higher rates (exemplified by higher dN and dN/dS) relative to cyt-aaRS, across mammals, birds, and Drosophila. While this pattern supports a model of compensatory evolution, the level at which a gene is expressed is a more general predictor of protein evolutionary rate. We find that gene expression level explains 10–56% of the variance in aaRS dN/dS, and that cyt-aaRS are more highly expressed in addition to having lower dN/dS values relative to mt-aaRS, consistent with more highly expressed genes being more evolutionarily constrained. Furthermore, we find no evidence of positive selection acting on either class of aaRS protein, as would be expected under a model of compensatory evolution. Nevertheless, the signature of faster mt-aaRS evolution persists in mammalian, but not bird or Drosophila, lineages after controlling for gene expression, suggesting some additional effect of compensatory evolution for mammalian mt-aaRS. We conclude that gene expression is the strongest factor governing differential amino acid substitution rates in proteins interacting with mitochondrial versus cytoplasmic factors, with important differences in mt-aaRS molecular evolution among taxonomic groups.

Rational design and directed evolution of a bacterial-type glutaminyl-tRNA synthetase precursor

Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNAGln is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNAGln, and an amidotransferase converts Glu-tRNAGln to Gln-tRNAGln. The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacylating Glu onto tRNAGln. It was proposed that GluRS2 is evolving into a bacterial-type GlnRS. Herein, we have combined rational design and directed evolution approaches to test this hypothesis. We show that, in contrast to wild-type (WT) GlnRS2, an engineered enzyme variant (M110) with seven amino acid changes is able to rescue growth of the temperature-sensitive Escherichia coli glnS strain UT172 at its non-permissive temperature. In vitro kinetic analyses reveal that WT GluRS2 selectively acylates Glu over Gln, whereas M110 acylates Gln 4-fold more efficiently than Glu. In addition, M110 hydrolyzes adenosine triphosphate 2.5-fold faster in the presence of Glu than Gln, suggesting that an editing activity has evolved in this variant to discriminate against Glu. These data imply that GluRS2 is a few steps away from evolving into a GlnRS and provides a paradigm for studying aminoacyl-tRNA synthetase evolution using directed engineering approaches.

Synthetase polyspecificity as a tool to modulate protein function

The site-specific incorporation of unnatural amino acids (UAAs) into proteins in bacteria is made possible by the evolution of aminoacyl-tRNA synthetases that selectively recognize and aminoacylate the amino acid of interest. Recently we have discovered that some of the previously evolved aaRSs display a degree of polyspecificity and are capable of recognizing multiple UAAs. Herein we report the polyspecificity of an aaRS evolved to encode a comarin containing amino acid. This polyspecificity was then exploited to introduce several UAAs into the fluorophore of GFP, altering its photophysical properties.

Incorporation of Fluorotyrosines into Ribonucleotide Reductase Using an Evolved, Polyspecific Aminoacyl-tRNA Synthetase

Tyrosyl radicals (Y·s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (F(n)Ys, n = 1-4) are promising tools for studying the mechanism of Y· formation and reactivity, as their pK(a) values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F(3)Y) and demonstrate their ability to charge an orthogonal tRNA with a series of F(n)Ys while maintaining high specificity over Y. An evolved aaRS is then used to incorporate F(n)Ys site-specifically into the two subunits (α2 and β2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y·s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with F(n)Y(s), and the resulting proteins are isolated in good yields. F(n)Ys incorporated at position 122 of β2, the site of a stable Y· in wild-type RNR, generate long-lived F(n)Y·s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y(122)(2,3,5)F(3)Y-β2 is energetically and/or conformationally modulated in such a way that the enzyme retains its activity but a new on-pathway Y· can accumulate. The distinct EPR properties of the 2,3,5-F(3)Y· facilitate spectral subtractions that make detection and identification of new Y·s straightforward.

Multilevel populations and the evolution of antibiotic resistance through horizontal gene transfer

Horizontal gene transfer (HGT) can create diversity in the genetic repertoire of a lineage. Successful gene transfer likely occurs more frequently between more closely related organisms, leading to the formation of higher-level exchange groups that in some respects are comparable to single-species populations. Genes that appear fixed in a single species can be replaced through distant homologs or iso-functional analogs acquired through HGT. These genes may originate from other species or they may be acquired by an individual strain from the species pan-genome. Because of their similarity to alleles in a population, we label these gene variants that are exchanged between related species as homeoalleles. In a case study, we show that biased gene transfer plays an important role in the evolution of aminoacyl-tRNA synthetases (aaRS). Many microorganisms make use of these genes against naturally occurring antibiotics. We suggest that the resistance against naturally occurring antibiotics is the likely driving force behind the frequent switching between divergent aaRS types and the reason for the maintenance of these homeoalleles in higher-level exchange groups. Resistance to naturally occurring antibiotics may lead to the maintenance of different types of aminoacyl-tRNA synthetases in Bacteria through gene transfer.

One plasmid selection system for the rapid evolution of aminoacyl-tRNA synthetases

We have developed a rapid, straightforward, one plasmid dual positive/negative selection system for the evolution of aminoacyl-tRNA synthetases with altered specificities in Escherichia coli. This system utilizes an amber stop codon containing chloramphenicol acetyltransferase/uracil phosphoribosyltransferase fusion gene. We demonstrate the utility of the system by identifying a variant of the Methanococcus jannaschii tyrosyl synthetase from a library of 10 9 variants that selectively incorporates para-iodophenylalanine in response to an amber stop codon.

Aminoacyl-tRNA synthetase classes and groups in prokaryotes

Knowledge on the evolution of aminoacyl-tRNA synthetases is crucial to studies on the origins of life. The relationships between the different aminoacyl-tRNA synthetase specificities in prokaryotic organisms are studied in this work. We reconstructed the ancestor sequences and the phylogenetic relationships utilizing the Maximum Likelihood method. The results suggest that in class I the evolution of the N-terminal segment was strongly influenced by the amino acid hydropathy in both domains of prokaryotes. The results for the C-terminal segments of class I were different in the two domains, indicating that its evolution was strongly influenced by the specific types of tRNA modification in each domain. The class II groups in Archaea were more heterogeneous with respect to the hydropathy of amino acids, indicating the interference of other influences. In bacteria, the configuration was also complex but the overall consensual division in two groups was maintained, group IIa forming a single branch with the five hydroapathetic amino acid specificities and group IIb containing the specificities for the moderately hydrophobic together with the hydrophilic amino acids. It is indicated that the aminoacyl-tRNA synthetase in both domains were subjected to different selective forces in diverse parts of the proteins, resulting in complex phylogenetic patterns.

Aminoacyl-tRNA synthetases: a complex system beyond protein synthesis

Aminoacyl-tRNA synthetases (ARSs) are enzymes that translate the genetic code by adding amino acids to their cognate transfer RNAs (tRNA). Aminoacylated tRNAs can then be used by the ribosome to decode mRNA. The essential role of ARSs was established in the 1960s, during the golden era of molecular biology that led to the discovery of the genetic code. The canonical role of these enzymes is now described in all textbooks. Remarkably, however, interest in ARS function continues to grow as new and unexpected functions are discovered for these enzymes, for tRNA, and for RNA in general. This article describes current progress in the field of ARS research, summarizes current thinking about the evolution of ARSs, introduces the readers to the many facets of cellular biology in which ARSs play an important role, and discusses the biotechnological applications derived from these studies.