The Roots of Genetic Coding in Aminoacyl-tRNA Synthetase Duality.

Codon-dependent translation underlies genetics and phylogenetic inferences, but its origins pose two challenges. Prevailing narratives cannot account for the fact that aminoacyl-tRNA synthetases (aaRSs), which translate the genetic code, must collectively enforce the rules used to assemble themselves. Nor can they explain how specific assignments arose from rudimentary differentiation between ancestral aaRSs and corresponding transfer RNAs (tRNAs). Experimental deconstruction of the two aaRS superfamilies created new experimental tools with which to analyze the emergence of the code. Amino acid and tRNA substrate recognition are linked to phase transfer free energies of amino acids and arise largely from aaRS class-specific differences in secondary structure. Sensitivity to protein folding rules endowed ancestral aaRS-tRNA pairs with the feedback necessary to rapidly compare alternative genetic codes and coding sequences. These and other experimental data suggest that the aaRS bidirectional genetic ancestry stabilized the differentiation and interdependence required to initiate and elaborate the genetic coding table.

Enzymic recognition of amino acids drove the evolution of primordial genetic codes.

How genetic information gained its exquisite control over chemical processes needed to build living cells remains an enigma. Today, the aminoacyl-tRNA synthetases (AARS) execute the genetic codes in all living systems. But how did the AARS that emerged over three billion years ago as low-specificity, protozymic forms then spawn the full range of highly-specific enzymes that distinguish between 22 diverse amino acids? A phylogenetic reconstruction of extant AARS genes, enhanced by analysing modular acquisitions, reveals six AARS with distinct bacterial, archaeal, eukaryotic, or organellar clades, resulting in a total of 36 families of AARS catalytic domains. Small structural modules that differentiate one AARS family from another played pivotal roles in discriminating between amino acid side chains, thereby expanding the genetic code and refining its precision. The resulting model shows a tendency for less elaborate enzymes, with simpler catalytic domains, to activate amino acids that were not synthesised until later in the evolution of the code. The most probable evolutionary route for an emergent amino acid type to establish a place in the code was by recruiting older, less specific AARS, rather than adapting contemporary lineages. This process, retrofunctionalisation, differs from previously described mechanisms through which amino acids would enter the code.

Primordial aminoacyl-tRNA synthetases preferred minihelices to full-length tRNA.

Aminoacyl-tRNA synthetases (AARS) and tRNAs translate the genetic code in all living cells. Little is known about how their molecular ancestors began to enforce the coding rules for the expression of their own genes. Schimmel et al. proposed in 1993 that AARS catalytic domains began by reading an 'operational' code in the acceptor stems of tRNA minihelices. We show here that the enzymology of an AARS urzyme•TΨC-minihelix cognate pair is a rich in vitro realization of that idea. The TΨC-minihelixLeu is a very poor substrate for full-length Leucyl-tRNA synthetase. It is a superior RNA substrate for the corresponding urzyme, LeuAC. LeuAC active-site mutations shift the choice of both amino acid and RNA substrates. AARS urzyme•minihelix cognate pairs are thus small, pliant models for the ancestral decoding hardware. They are thus an ideal platform for detailed experimental study of the operational RNA code.

Domain acquisition by class I aminoacyl-tRNA synthetase urzymes coordinated the catalytic functions of HVGH and KMSKS motifs.

Leucyl-tRNA synthetase (LeuRS) is a Class I aminoacyl-tRNA synthetase (aaRS) that synthesizes leucyl-tRNAleu for codon-directed protein synthesis. Two signature sequences, HxGH and KMSKS help stabilize transition-states for amino acid activation and tRNA aminoacylation by all Class I aaRS. Separate alanine mutants of each signature, together with the double mutant, behave in opposite ways in Pyrococcus horikoshii LeuRS and the 129-residue urzyme ancestral model generated from it (LeuAC). Free energy coupling terms, Δ(ΔG‡), for both reactions are large and favourable for LeuRS, but unfavourable for LeuAC. Single turnover assays with 32Pα-ATP show correspondingly different internal products. These results implicate domain motion in catalysis by full-length LeuRS. The distributed thermodynamic cycle of mutational changes authenticates LeuAC urzyme catalysis far more convincingly than do single point mutations. Most importantly, the evolutionary gain of function induced by acquiring the anticodon-binding (ABD) and multiple insertion modules in the catalytic domain appears to be to coordinate the catalytic function of the HxGH and KMSKS signature sequences. The implication that backbone elements of secondary structures achieve a major portion of the overall transition-state stabilization by LeuAC is also consistent with coevolution of the genetic code and metabolic pathways necessary to produce histidine and lysine sidechains.

A Leucyl-tRNA Synthetase Urzyme: Authenticity of tRNA Synthetase Catalytic Activities and Promiscuous Phosphorylation of Leucyl-5'AMP.

Aminoacyl-tRNA synthetase (aaRS)/tRNA cognate pairs translate the genetic code by synthesizing specific aminoacyl-tRNAs that are assembled on messenger RNA by the ribosome. Deconstruction of the two distinct aaRS superfamilies (Classes) has provided conceptual and experimental models for their early evolution. Urzymes, containing ~120-130 amino acids excerpted from regions where genetic coding sequence complementarities have been identified, are key experimental models motivated by the proposal of a single bidirectional ancestral gene. Previous reports that Class I and Class II urzymes accelerate both amino acid activation and tRNA aminoacylation have not been extended to other synthetases. We describe a third urzyme (LeuAC) prepared from the Class IA Pyrococcus horikoshii leucyl-tRNA synthetase. We adduce multiple lines of evidence for the authenticity of its catalysis of both canonical reactions, amino acid activation and tRNALeu aminoacylation. Mutation of the three active-site lysine residues to alanine causes significant, but modest reduction in both amino acid activation and aminoacylation. LeuAC also catalyzes production of ADP, a non-canonical enzymatic function that has been overlooked since it first was described for several full-length aaRS in the 1970s. Structural data suggest that the LeuAC active site accommodates two ATP conformations that are prominent in water but rarely seen bound to proteins, accounting for successive, in situ phosphorylation of the bound leucyl-5'AMP phosphate, accounting for ADP production. This unusual ATP consumption regenerates the transition state for amino acid activation and suggests, in turn, that in the absence of the editing and anticodon-binding domains, LeuAC releases leu-5'AMP unusually slowly, relative to the two phosphorylation reactions.

Multidimensional Phylogenetic Metrics Identify Class I Aminoacyl-tRNA Synthetase Evolutionary Mosaicity and Inter-Modular Coupling.

The role of aminoacyl-tRNA synthetases (aaRS) in the emergence and evolution of genetic coding poses challenging questions concerning their provenance. We seek evidence about their ancestry from curated structure-based multiple sequence alignments of a structurally invariant "scaffold" shared by all 10 canonical Class I aaRS. Three uncorrelated phylogenetic metrics-mutation frequency, its uniformity, and row-by-row cladistic congruence-imply that the Class I scaffold is a mosaic assembled from successive genetic sources. Metrics for different modules vary in accordance with their presumed functionality. Sequences derived from the ATP- and amino acid- binding sites exhibit specific two-way coupling to those derived from Connecting Peptide 1, a third module whose metrics suggest later acquisition. The data help validate: (i) experimental fragmentations of the canonical Class I structure into three partitions that retain catalytic activities in proportion to their length; and (ii) evidence that the ancestral Class I aaRS gene also encoded a Class II ancestor in frame on the opposite strand. A 46-residue Class I "protozyme" roots the Class I tree prior to the adaptive radiation of the Rossmann dinucleotide binding fold that refined substrate discrimination. Such rooting implies near simultaneous emergence of genetic coding and the origin of the proteome, resolving a conundrum posed by previous inferences that Class I aaRS evolved after the genetic code had been implemented in an RNA world. Further, pinpointing discontinuous enhancements of aaRS fidelity establishes a timeline for the growth of coding from a binary amino acid alphabet.

Escapement mechanisms: Efficient free energy transduction by reciprocally-coupled gating.

Conversion of the free energy of NTP hydrolysis efficiently into mechanical work and/or information by transducing enzymes sustains living systems far from equilibrium, and so has been of interest for many decades. Detailed molecular mechanisms, however, remain puzzling and incomplete. We previously reported that catalysis of tryptophan activation by tryptophanyl-tRNA synthetase, TrpRS, requires relative domain motion to re-position the catalytic Mg2+ ion, noting the analogy between that conditional hydrolysis of ATP and the escapement mechanism of a mechanical clock. The escapement allows the time-keeping mechanism to advance discretely, one gear at a time, if and only if the pendulum swings, thereby converting energy from the weight driving the pendulum into rotation of the hands. Coupling of catalysis to domain motion, however, mimics only half of the escapement mechanism, suggesting that domain motion may also be reciprocally coupled to catalysis, completing the escapement metaphor. Computational studies of the free energy surface restraining the domain motion later confirmed that reciprocal coupling: the catalytic domain motion is thermodynamically unfavorable unless the PPi product is released from the active site. These two conditional phenomena-demonstrated together only for the TrpRS mechanism-function as reciprocally-coupled gates. As we and others have noted, such an escapement mechanism is essential to the efficient transduction of NTP hydrolysis free energy into other useful forms of mechanical or chemical work and/or information. Some implementation of both gating mechanisms-catalysis by domain motion and domain motion by catalysis-will thus likely be found in many other systems.

Hierarchical groove discrimination by Class I and II aminoacyl-tRNA synthetases reveals a palimpsest of the operational RNA code in the tRNA acceptor-stem bases.

Class I and II aaRS recognition of opposite grooves was likely among the earliest determinants fixed in the tRNA acceptor stem bases. A new regression model identifies those determinants in bacterial tRNAs. Integral coefficients relate digital dependent to independent variables with perfect agreement between observed and calculated grooves for all twenty isoaccepting tRNAs. Recognition is mediated by the Discriminator base 73, the first base pair, and base 2 of the acceptor stem. Subsets of these coefficients also identically compute grooves recognized by smaller numbers of aaRS. Thus, the model is hierarchical, suggesting that new rules were added to pre-existing ones as new amino acids joined the coding alphabet. A thermodynamic rationale for the simplest model implies that Class-dependent aaRS secondary structures exploited differential tendencies of the acceptor stem to form the hairpin observed in Class I aaRS•tRNA complexes, enabling the earliest groove discrimination. Curiously, groove recognition also depends explicitly on the identity of base 2 in a manner consistent with the middle bases of the codon table, confirming a hidden ancestry of codon-anticodon pairing in the acceptor stem. That, and the lack of correlation with anticodon bases support prior productive coding interaction of tRNA minihelices with proto-mRNA.

Impedance Matching and the Choice Between Alternative Pathways for the Origin of Genetic Coding.

We recently observed that errors in gene replication and translation could be seen qualitatively to behave analogously to the impedances in acoustical and electronic energy transducing systems. We develop here quantitative relationships necessary to confirm that analogy and to place it into the context of the minimization of dissipative losses of both chemical free energy and information. The formal developments include expressions for the information transferred from a template to a new polymer, Iσ; an impedance parameter, Z; and an effective alphabet size, neff; all of which have non-linear dependences on the fidelity parameter, q, and the alphabet size, n. Surfaces of these functions over the {n,q} plane reveal key new insights into the origin of coding. Our conclusion is that the emergence and evolutionary refinement of information transfer in biology follow principles previously identified to govern physical energy flows, strengthening analogies (i) between chemical self-organization and biological natural selection, and (ii) between the course of evolutionary trajectories and the most probable pathways for time-dependent transitions in physics. Matching the informational impedance of translation to the four-letter alphabet of genes uncovers a pivotal role for the redundancy of triplet codons in preserving as much intrinsic genetic information as possible, especially in early stages when the coding alphabet size was small.

Interdependence, Reflexivity, Fidelity, Impedance Matching, and the Evolution of Genetic Coding.

Genetic coding is generally thought to have required ribozymes whose functions were taken over by polypeptide aminoacyl-tRNA synthetases (aaRS). Two discoveries about aaRS and their interactions with tRNA substrates now furnish a unifying rationale for the opposite conclusion: that the key processes of the Central Dogma of molecular biology emerged simultaneously and naturally from simple origins in a peptide•RNA partnership, eliminating the epistemological utility of a prior RNA world. First, the two aaRS classes likely arose from opposite strands of the same ancestral gene, implying a simple genetic alphabet. The resulting inversion symmetries in aaRS structural biology would have stabilized the initial and subsequent differentiation of coding specificities, rapidly promoting diversity in the proteome. Second, amino acid physical chemistry maps onto tRNA identity elements, establishing reflexive, nanoenvironmental sensing in protein aaRS. Bootstrapping of increasingly detailed coding is thus intrinsic to polypeptide aaRS, but impossible in an RNA world. These notions underline the following concepts that contradict gradual replacement of ribozymal aaRS by polypeptide aaRS: 1) aaRS enzymes must be interdependent; 2) reflexivity intrinsic to polypeptide aaRS production dynamics promotes bootstrapping; 3) takeover of RNA-catalyzed aminoacylation by enzymes will necessarily degrade specificity; and 4) the Central Dogma's emergence is most probable when replication and translation error rates remain comparable. These characteristics are necessary and sufficient for the essentially de novo emergence of a coupled gene-replicase-translatase system of genetic coding that would have continuously preserved the functional meaning of genetically encoded protein genes whose phylogenetic relationships match those observed today.

Class I and II aminoacyl-tRNA synthetase tRNA groove discrimination created the first synthetase-tRNA cognate pairs and was therefore essential to the origin of genetic coding.

The genetic code likely arose when a bidirectional gene replicating as a quasi-species began to produce ancestral aminoacyl-tRNA synthetases (aaRS) capable of distinguishing between two distinct sets of amino acids. The synthetase class division therefore necessarily implies a mechanism by which the two ancestral synthetases could also discriminate between two different kinds of tRNA substrates. We used regression methods to uncover the possible patterns of base sequences capable of such discrimination and find that they appear to be related to thermodynamic differences in the relative stabilities of a hairpin necessary for recognition of tRNA substrates by Class I aaRS. The thermodynamic differences appear to be exploited by secondary structural differences between models for the ancestral aaRS called synthetase Urzymes and reinforced by packing of aromatic amino acid side chains against the nonpolar face of the ribose of A76 if and only if the tRNA CCA sequence forms a hairpin. The patterns of bases 1, 2, and 73 and stabilization of the hairpin by structural complementarity with Class I, but not Class II, aaRS Urzymes appear to be necessary and sufficient to have enabled the generation of the first two aaRS-tRNA cognate pairs, and the launch of a rudimentary binary genetic coding related recognizably to contemporary cognate pairs. As a consequence, it seems likely that nonrandom aminoacylation of tRNAs preceded the advent of the tRNA anticodon stem-loop. Consistent with this suggestion, coding rules in the acceptor-stem bases also reveal a palimpsest of the codon-anticodon interaction, as previously proposed. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1088-1098, 2019.

tRNA acceptor stem and anticodon bases form independent codes related to protein folding.

Aminoacyl-tRNA synthetases recognize tRNA anticodon and 3' acceptor stem bases. Synthetase Urzymes acylate cognate tRNAs even without anticodon-binding domains, in keeping with the possibility that acceptor stem recognition preceded anticodon recognition. Representing tRNA identity elements with two bits per base, we show that the anticodon encodes the hydrophobicity of each amino acid side-chain as represented by its water-to-cyclohexane distribution coefficient, and this relationship holds true over the entire temperature range of liquid water. The acceptor stem codes preferentially for the surface area or size of each side-chain, as represented by its vapor-to-cyclohexane distribution coefficient. These orthogonal experimental properties are both necessary to account satisfactorily for the exposed surface area of amino acids in folded proteins. Moreover, the acceptor stem codes correctly for ß-branched and carboxylic acid side-chains, whereas the anticodon codes for a wider range of such properties, but not for size or ß-branching. These and other results suggest that genetic coding of 3D protein structures evolved in distinct stages, based initially on the size of the amino acid and later on its compatibility with globular folding in water.

Temperature dependence of amino acid hydrophobicities.

The hydrophobicities of the 20 common amino acids are reflected in their tendencies to appear in interior positions in globular proteins and in deeply buried positions of membrane proteins. To determine whether these relationships might also have been valid in the warm surroundings where life may have originated, we examined the effect of temperature on the hydrophobicities of the amino acids as measured by the equilibrium constants for transfer of their side-chains from neutral solution to cyclohexane (K(w > c)). The hydrophobicities of most amino acids were found to increase with increasing temperature. Because that effect is more pronounced for the more polar amino acids, the numerical range of K(w > c) values decreases with increasing temperature. There are also modest changes in the ordering of the more polar amino acids. However, those changes are such that they would have tended to minimize the otherwise disruptive effects of a changing thermal environment on the evolution of protein structure. Earlier, the genetic code was found to be organized in such a way that--with a single exception (threonine)--the side-chain dichotomy polar/nonpolar matches the nucleic acid base dichotomy purine/pyrimidine at the second position of each coding triplet at 25 °C. That dichotomy is preserved at 100 °C. The accessible surface areas of amino acid side-chains in folded proteins are moderately correlated with hydrophobicity, but when free energies of vapor-to-cyclohexane transfer (corresponding to size) are taken into consideration, a closer relationship becomes apparent.

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based.

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (Km ∼ 2 µm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg(2+) ion in a manner that allows it to assist in transition state stabilization. The Mg(2+) ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg(2+)·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg(2+) coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg(2+). Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to HcTrpRS.

Coding of Class I and II Aminoacyl-tRNA Synthetases.

The aminoacyl-tRNA synthetases and their cognate transfer RNAs translate the universal genetic code. The twenty canonical amino acids are sufficiently diverse to create a selective advantage for dividing amino acid activation between two distinct, apparently unrelated superfamilies of synthetases, Class I amino acids being generally larger and less polar, Class II amino acids smaller and more polar. Biochemical, bioinformatic, and protein engineering experiments support the hypothesis that the two Classes descended from opposite strands of the same ancestral gene. Parallel experimental deconstructions of Class I and II synthetases reveal parallel losses in catalytic proficiency at two novel modular levels-protozymes and Urzymes-associated with the evolution of catalytic activity. Bi-directional coding supports an important unification of the proteome; affords a genetic relatedness metric-middle base-pairing frequencies in sense/antisense alignments-that probes more deeply into the evolutionary history of translation than do single multiple sequence alignments; and has facilitated the analysis of hitherto unknown coding relationships in tRNA sequences. Reconstruction of native synthetases by modular thermodynamic cycles facilitated by domain engineering emphasizes the subtlety associated with achieving high specificity, shedding new light on allosteric relationships in contemporary synthetases. Synthetase Urzyme structural biology suggests that they are catalytically-active molten globules, broadening the potential manifold of polypeptide catalysts accessible to primitive genetic coding and motivating revisions of the origins of catalysis. Finally, bi-directional genetic coding of some of the oldest genes in the proteome places major limitations on the likelihood that any RNA World preceded the origins of coded proteins.

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene.

Aminoacyl-tRNA synthetases (aaRS) catalyze both chemical steps that translate the universal genetic code. Rodin and Ohno offered an explanation for the existence of two aaRS classes, observing that codons for the most highly conserved Class I active-site residues are anticodons for corresponding Class II active-site residues. They proposed that the two classes arose simultaneously, by translation of opposite strands from the same gene. We have characterized wild-type 46-residue peptides containing ATP-binding sites of Class I and II synthetases and those coded by a gene designed by Rosetta to encode the corresponding peptides on opposite strands. Catalysis by WT and designed peptides is saturable, and the designed peptides are sensitive to active-site residue mutation. All have comparable apparent second-order rate constants 2.9-7.0E-3 M(-1) s(-1) or ∼750,000-1,300,000 times the uncatalyzed rate. The activities of the two complementary peptides demonstrate that the unique information in a gene can have two functional interpretations, one from each complementary strand. The peptides contain phylogenetic signatures of longer, more sophisticated catalysts we call Urzymes and are short enough to bridge the gap between them and simpler uncoded peptides. Thus, they directly substantiate the sense/antisense coding ancestry of Class I and II aaRS. Furthermore, designed 46-mers achieve similar catalytic proficiency to wild-type 46-mers by significant increases in both kcat and Km values, supporting suggestions that the earliest peptide catalysts activated ATP for biosynthetic purposes.

tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry.

The universal genetic code is a translation table by which nucleic acid sequences can be interpreted as polypeptides with a wide range of biological functions. That information is used by aminoacyl-tRNA synthetases to translate the code. Moreover, amino acid properties dictate protein folding. We recently reported that digital correlation techniques could identify patterns in tRNA identity elements that govern recognition by synthetases. Our analysis, and the functionality of truncated synthetases that cannot recognize the tRNA anticodon, support the conclusion that the tRNA acceptor stem houses an independent code for the same 20 amino acids that likely functioned earlier in the emergence of genetics. The acceptor-stem code, related to amino acid size, is distinct from a code in the anticodon that is related to amino acid polarity. Details of the acceptor-stem code suggest that it was useful in preserving key properties of stereochemically-encoded peptides that had developed the capacity to interact catalytically with RNA. The quantitative embedding of the chemical properties of amino acids into tRNA bases has implications for the origins of molecular biology.

Urzymology: experimental access to a key transition in the appearance of enzymes.

Urzymes are catalysts derived from invariant cores of protein superfamilies. Urzymes from both aminoacyl-tRNA synthetase classes possess sophisticated catalytic mechanisms: pre-steady state bursts, significant transition-state stabilization of both amino acid activation, and tRNA acylation. However, they have insufficient specificity to ensure a fully developed genetic code, suggesting that they participated in synthesizing statistical proteins. They represent a robust experimental platform from which to articulate and test hypotheses both about their own ancestors and about how they, in turn, evolved into modern enzymes. They help reshape numerous paradigms from the RNA World hypothesis to protein structure databases and allostery.

Enhanced amino acid selection in fully evolved tryptophanyl-tRNA synthetase, relative to its urzyme, requires domain motion sensed by the D1 switch, a remote dynamic packing motif.

We previously showed (Li, L., and Carter, C. W., Jr. (2013) J. Biol. Chem. 288, 34736-34745) that increased specificity for tryptophan versus tyrosine by contemporary Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) over that of TrpRS Urzyme results entirely from coupling between the anticodon-binding domain and an insertion into the Rossmann-fold known as Connecting Peptide 1. We show that this effect is closely related to a long range catalytic effect, in which side chain repacking in a region called the D1 Switch, accounts fully for the entire catalytic contribution of the catalytic Mg(2+) ion. We report intrinsic and higher order interaction effects on the specificity ratio, (kcat/Km)Trp/(kcat/Km)Tyr, of 15 combinatorial mutants from a previous study (Weinreb, V., Li, L., and Carter, C. W., Jr. (2012) Structure 20, 128-138) of the catalytic role of the D1 Switch. Unexpectedly, the same four-way interaction both activates catalytic assist by Mg(2+) ion and contributes -4.4 kcal/mol to the free energy of the specificity ratio. A minimum action path computed for the induced-fit and catalytic conformation changes shows that repacking of the four residues precedes a decrease in the volume of the tryptophan-binding pocket. We suggest that previous efforts to alter amino acid specificities of TrpRS and glutaminyl-tRNA synthetase (GlnRS) by mutagenesis without extensive, modular substitution failed because mutations were incompatible with interdomain motions required for catalysis.

Full implementation of the genetic code by tryptophanyl-tRNA synthetase requires intermodular coupling.

Tryptophanyl-tRNA Synthetase (TrpRS) Urzyme (fragments A and C), a 130-residue construct containing only secondary structures positioning the HIGH and KMSKS active site signatures and the specificity helix, accelerates tRNA(Trp) aminoacylation with ∼10-fold specificity toward tryptophan, relative to structurally related tyrosine. We proposed that including the 76-residue connecting peptide 1 insertion (Fragment B) might enhance tryptophan affinity and hence amino acid specificity, because that subdomain constrains the orientation of the specificity helix. We test that hypothesis by characterizing two new constructs: the catalytic domain (fragments A-C) and the Urzyme supplemented with the anticodon-binding domain (fragments A, C, and D). The three constructs, together with the full-length enzyme (fragments A-D), comprise a factorial experiment from which we deduce individual and combined contributions of the two modules to the steady-state kinetics parameters for tryptophan-dependent (32)PPi exchange, specificity for tryptophan versus tyrosine, and aminoacylation of tRNA(Trp). Factorial design directly measures the energetic coupling between the two more recent modules in the contemporary enzyme and demonstrates its functionality. Combining the TrpRS Urzyme individually in cis with each module affords an analysis of long term evolution of amino acid specificity and tRNA aminoacylation, both essential for expanding the genetic code. Either module significantly enhances tryptophan activation but unexpectedly eliminates amino acid specificity for tryptophan, relative to tyrosine, and significantly reduces tRNA aminoacylation. Exclusive dependence of both enhanced functionalities of full-length TrpRS on interdomain coupling energies between the two new modules argues that independent recruitment of connecting peptide 1 and the anticodon-binding domain during evolutionary development of Urzymes would have entailed significant losses of fitness.