Rate Of Aminoacylation Research Articles

A tRNAModification-based strategy for Identifying amiNo acid Overproducers (AMINO)

Amino acids have a multi-billion-dollar market with rising demand, prompting the development of high-performance microbial factories. However, a general screening strategy applicable to all proteinogenic and non-proteinogenic amino acids is still lacking. Modification of the critical structure of tRNA could decrease the aminoacylation level of tRNA catalyzed by aminoacyl-tRNA synthetases. Involved in a two-substrate sequential reaction, amino acids with increased concentration could elevate the reduced aminoacylation rate caused by specific tRNA modification. Here, we developed a selection system for overproducers of specific amino acids using corresponding engineered tRNAs and marker genes. As a proof-of-concept, overproducers of five amino acids such as L-tryptophan were screened out by growth-based and/or fluorescence-activated cell sorting (FACS)-based screening from random mutation libraries of Escherichia coli and Corynebacterium glutamicum, respectively. This study provided a universal strategy that could be applied to screen overproducers of proteinogenic and non-proteinogenic amino acids in amber-stop-codon-recoded or non-recoded hosts.

Role of two aminoacyl-tRNA synthetase associated proteins (Endothelial Monocyte Activating Polypeptides 1 and 2) of Leishmania donovani in chemotaxis of human monocytes.

Visceral leishmaniasis is caused by the protozoan parasite Leishmania donovani. It is a fatal form of leishmaniasis prevalent in Indian subcontinent. Since there are no human licensed vaccines available for leishmaniasis, chemotherapeutic drugs remain the only means for combating parasitic infections. We have earlier identified a total of 26 amino-acyl tRNA synthetases (aaRS) along with five stand-alone editing domains and two aaRS-associated proteins in Leishmania donovani. In addition to their canonical role of tRNA aminoacylation, aaRS have been involved in novel functions by acquiring novel domains during evolution. The aaRS-associated proteins have been reported to be analogous to a human cytokine, EMAP II, as they possess a modified version of the heptapeptide motif responsible for the cytokine activity. In this manuscript, we report the characterization of two L. donovani aminoacyl-tRNA synthetase associated proteins which showed a human chemokine like activity. Both the proteins, L. donovani EMAP-1 and EMAP-2, possess a modified form of the heptapeptide motif, which is responsible for cytokine activity in human EMAP-2. LdEMAP-1 and LdEMAP-2 were cloned, expressed, and purified. Both LdEMAP-1 and LdEMAP-2 proteins in the promastigote stage were found to be localized in cytoplasm as confirmed by immunofluorescence. In case of L. donovani infected human THP-1 derived macrophages, secretion of LdEMAP-1 and LdEMAP-2 proteins in the cytosol of the macrophages was observed. The role of LdEMAP-1 and LdEMAP-2 in the aminoacylation of rLdTyrRS was also tested and LdEMAP-2 but not LdEMAP-1 increased the rate of aminoacylation of tyrosyl tRNA synthetase (rLdTyrRS). L. donovani EMAP-1 and EMAP-2 proteins managed to exhibit the capability of attracting human origin cells as determined by chemotaxis assay, and also were able to induce the secretion of cytokines from macrophages like their human counterpart (EMAP II). Our working hypothesis is that both of these proteins might be involved in helping the parasite to establish the infection within the host.

Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

Pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl have been extensively used for genetic-code expansion. A Methanosarcina mazei PylRS mutant bearing the Y306A and Y384F mutations (PylRS(Y306A/Y384F)) encodes various bulky non-natural lysine derivatives by UAG. In this study, we examined how PylRS(Y306A/Y384F) recognizes many amino acids. Among 17 non-natural lysine derivatives, Nɛ-(benzyloxycarbonyl)lysine (ZLys) and 10 ortho/meta/para-substituted ZLys derivatives were efficiently ligated to tRNAPyl and were incorporated into proteins by PylRS(Y306A/Y384F). We determined crystal structures of 14 non-natural lysine derivatives bound tothe PylRS(Y306A/Y384F) catalytic fragment. The meta- and para-substituted ZLys derivatives are snugly accommodated in the productive mode. In contrast, ZLys and the unsubstituted or ortho-substituted ZLys derivatives exhibited an alternative binding mode in addition to the productive mode. PylRS(Y306A/Y384F) displayed a high aminoacylation rate for ZLys, indicating that the double-binding mode minimally affects aminoacylation. These precise substrate recognition mechanisms by PylRS(Y306A/Y384F) may facilitate the structure-based design of novel non-natural amino acids.

Structural Basis for Genetic-Code Expansion with Various Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

Pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl have been extensively used for genetic-code expansion. A Methanosarcina mazei PylRS mutant bearing the Y306A and Y384F mutations (PylRS(Y306A/Y384F)) encodes various bulky non-natural lysine derivatives by UAG. In this study, we examined how PylRS(Y306A/Y384F) recognizes many amino acids. Among 17 non-natural lysine derivatives, Ne-(benzyloxycarbonyl)lysine (ZLys) and 10 ortho/meta/para-substituted ZLys derivatives were efficiently tRNAPyl ligated and protein incorporated by PylRS(Y306A/Y384F). We determined crystal structures of 14 non-natural lysine derivatives bound to the PylRS(Y306A/Y384F) catalytic fragment. The meta- and para-substituted ZLys derivatives are snugly accommodated in the productive mode. In contrast, ZLys and the unsubstituted or ortho-substituted ZLys derivatives exhibited an alternative binding mode in addition to the productive mode. PylRS(Y306A/Y384F) displayed a high aminoacylation rate for ZLys, indicating that the double binding mode minimally affects aminoacylation. These precise substrate recognition mechanisms by PylRS(Y306A/Y384F) may facilitate the structure-based design of novel non-natural amino acids.

Aminoacyl Transfer Rate Dictates Choice of Editing Pathway in Threonyl-tRNA Synthetase

Aminoacyl-tRNA synthetases hydrolyze aminoacyl adenylates and aminoacyl-tRNAs formed from near-cognate amino acids, thereby increasing translational fidelity. The contributions of pre- and post-transfer editing pathways to the fidelity of Escherichia coli threonyl-tRNA synthetase (ThrRS) were investigated by rapid kinetics. In the pre-steady state, asymmetric activation of cognate threonine and noncognate serine was observed in the active sites of dimeric ThrRS, with similar rates of activation. In the absence of tRNA, seryl-adenylate was hydrolyzed 29-fold faster by the ThrRS catalytic domain than threonyl-adenylate. The rate of seryl transfer to cognate tRNA was only 2-fold slower than threonine. Experiments comparing the rate of ATP consumption to the rate of aminoacyl-tRNA(AA) formation demonstrated that pre-transfer hydrolysis contributes to proofreading only when the rate of transfer is slowed significantly. Thus, the relative contributions of pre- and post-transfer editing in ThrRS are subject to modulation by the rate of aminoacyl transfer.

A Disease-causing Point Mutation in Human Mitochondrial tRNAMet Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation

The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNA(Met) (hmtRNA(Met)) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNA(Met) with a C8. The hmtRNA(Met) serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U-->C mutation leads to a failure of the tRNA to respond conformationally to Mg(2+). This mutation results in a drastic disruption of the structure of the hmtRNA(Met), which significantly reduces its aminoacylation. The small fraction of hmtRNA(Met) that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg(2+), the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg(2+), the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg(2+)-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans.

Multiple mechanisms to ameliorate the fitness burden of mupirocin resistance in Salmonella typhimurium

We examined how the fitness costs of mupirocin resistance caused by mutations in the chromosomal isoleucyl-tRNA synthetase gene (ileS) can be ameliorated. Mupirocin-resistant mutants were isolated and four different, resistance-conferring point mutations in the chromosomal ileS gene were identified. Fifty independent lineages of the low-fitness, resistant mutants were serially passaged to evolve compensated mutants with increased fitness. In 34/50 of the evolved lineages, the increase in fitness resulted from additional point mutations in isoleucine tRNA synthetase (IleRS). Measurements in vitro of the kinetics of aminoacylation of wild-type and mutant enzymes showed that resistant IleRS had a reduced rate of aminoacylation due to altered interactions with both tRNAIle and ATP. The intragenic compensatory mutations improved IleRS kinetics towards the wild-type enzyme, thereby restoring bacterial fitness. Seven of the 16 lineages that lacked second-site compensatory mutations in ileS, showed an increase in ileS gene dosage, suggesting that an increased level of defective IleRS compensate for the decrease in aminoacylation activity. Our findings show that the fitness costs of ileS mutations conferring mupirocin resistance can be reduced by several types of mechanisms that may contribute to the stability of mupirocin resistance in clinical settings.

Distinct Kinetic Mechanisms of the Two Classes of Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases are divided into two classes based on both functional and structural criteria. Distinctions between the classes have heretofore been based on general features, such as the position of aminoacylation on the 3′-terminal tRNA ribose, and the topology and tRNA-binding orientation of the active-site protein fold. Here we show instead that transient burst kinetics provides a distinct mechanistic signature dividing the two classes of tRNA synthetases, and that this distinction has significant downstream effects on protein synthesis. Steady-state and transient kinetic analyses of class I CysRS and ValRS, and class II AlaRS and ProRS, reveal that class I tRNA synthetases are rate-limited by release of aminoacyl-tRNA, while class II synthetases are limited by a step prior to aminoacyl transfer. The tight aminoacyl-tRNA product binding by class I enzymes correlates with the ability of EF-Tu to form a ternary complex with class I but not class II synthetases, and the further capacity of this protein to enhance the rate of aminoacylation by class I synthetases. These results emphasize that the distinct mechanistic signatures of class I versus class II tRNA synthetases ensure rapid turnover of aminoacyl-tRNAs during protein synthesis.

A Substrate-Assisted Concerted Mechanism for Aminoacylation by a Class II Aminoacyl-tRNA Synthetase

Aminoacyl-tRNA synthetases (aaRS) join amino acids to their cognate transfer RNAs, establishing an essential coding relationship in translation. To investigate the mechanism of aminoacyl transfer in class II Escherichia coli histidyl-tRNA synthetase (HisRS), we devised a rapid quench assay. Under single turnover conditions with limiting tRNA, aminoacyl transfer proceeds at 18.8 s(-)(1), whereas in the steady state, the overall rate of aminoacylation is limited by amino acid activation to a rate of 3 s(-)(1). In vivo, this mechanism may serve to allow the size of amino acid pools and energy charge to control the rate of aminoacylation and thus protein synthesis. Aminoacyl transfer experiments using HisRS active site mutants and phosphorothioate-substituted adenylate showed that substitution of the nonbridging Sp oxygen of the adenylate decreased the transfer rate at least 10 000-fold, providing direct experimental evidence for the role of this group as a general base for the reaction. Other kinetic experiments revealed that the rate of aminoacyl transfer is independent of the interaction between the carboxyamide group of Gln127 and the alpha-carboxylate carbon, arguing against the formation of a tetrahedral intermediate during the aminoacyl transfer. These experiments support a substrate-assisted concerted mechanism for HisRS, a feature that may generalize to other aaRS, as well as the peptidyl transferase center.

A novel chemically and mechanically stable glycerol-based crosslinked polystyrene support for polypeptide synthesis: A comparative study with Merrifield resin

A novel chemically and mechanically stable polymer support for solid-phase peptide synthesis with excellent swelling performance in various organic solvents is described. The crosslinked polymer in its bead form was prepared by the aqueous suspension polymerization of the styrene and tri(propylene glycol) glycerolate diacrylate. The support is unique because the hydroxyl group is introduced into the polymer support in the polymerization step itself, which makes it highly cost effective. The hydroxyl functionality of the crosslinker in the polymer was used as a growth site for peptide synthesis, thus minimizing the incompatibility arising because of the styrene core as compared to styrene-based polymer supports currently used in polypeptide synthesis. The utility of the resin for solid-phase peptide synthesis was tested by the synthesis of a 19-residue peptide amide of the envelope region of hepatitis C viral polyprotein and was compared with Merrifield resin by the 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluoro phosphate/1-hydroxy benzatriazole method with a Rink amide linker. The molecular character of the polymer and the extent of crosslinking density on the dependence of the reactivity of the attached amino groups were investigated by a reactivity study of the amide bond formation of a model Val-Ala dipeptide. Enhanced swelling, the increased rate of aminoacylation, and the high purity of the peptides synthesized on the novel support highlighted the influence of the polarity of the solid support in the solid-phase synthesis. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 288–294, 2004

TRNA discrimination by T. thermophilus phenylalanyl-tRNA synthetase at the binding step.

The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs.

Transfer RNA Phe isoacceptors possess non-identical set of identity elements at high and low Mg 2+ concentration

Primary structures of phage T5- and Escherichia coli-encoded tRNA Phe are distinct at four out of 11 positions known as identity elements for E. coli phenylalanyl-tRNA synthetase (FRS). In order to reveal structural requirements for FRS recognition, aminoacylation of wild-type phage T5 tRNA Phe gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E. coli tRNA Phe gene transcript. The wild-type phage T5 transcript can be aminoacylated with the same catalytic efficiency as the E. coli counterpart. However, the maximal aminoacylation rate for T5 and E. coli transcripts was reached at different Mg 2+ concentrations: 4 and 15 mM, respectively. Aminoacylation assays with tRNA Phe mutants revealed that (i) phage transcripts with the substituted anticodon bases at positions 35 and 36 were efficient substrates for aminoacylation at 15 mM Mg 2+ but not at optimal 4 mM Mg 2+; (ii) any change of G34 in phage transcripts dramatically decreased the aminoacylation efficiency at both 4 and 15 mM Mg 2+ whereas G34A mutation in the E. coli transcript exhibits virtually no influence on aminoacylation rate at 15 mM Mg 2+; (iii) substitution of A20 with U in the phage transcript caused no significant change in the aminoacylation rate at both Mg 2+ concentrations; (iv) phage transcripts with double substitutions A20U+A35C and A20U+A36C were very poor substrates for FRS. Collectively, the results indicate the non-identical mode of tRNA Phe recognition by E. coli FRS at low and high Mg 2+ concentrations. Probably, along with identity elements, the local tRNA conformation is essential for recognition by FRS.

Small-molecule-substrate interactions with a self-aminoacylating ribozyme

A self-aminoacylating RNA catalyst is shown to carry out the chemistry required for turnover, being reacylated several times from aminoacyl-AMP with an unaltered rate, thereby meeting one definition of an enzyme. Furthermore, a newly applied gel electrophoresis assay suggests first order kinetics in RNA and saturation kinetics in the substrate aminoacyl-adenylate, implying a Michaelis complex. AMP is a competitive inhibitor, though phenylalanine is not detectably inhibitory, consistent with a Michaelis complex through the AMP moiety of phenylalanyl-adenylate substrate. This idea is supported by measurement of elevated acylation velocities with seryl and alanyl-adenylates. The rate of aminoacylation increases with pH, consistent with attack of a terminal ribose oxyanion on the carbonyl carbon atom of the adenylate.

Variant minihelix RNAs reveal sequence-specific recognition of the helical tRNA(Ser) acceptor stem by E.coli seryl-tRNA synthetase.

Aminoacylation rate determinations for a series of variant RNA minihelix substrates revealed that Escherichia coli seryl-tRNA synthetase (SerRS) recognizes the 1--72 through 5--68 base pairs of the E.coli tRNA(Ser) acceptor stem with the major recognition elements clustered between positions 2--71 and 4--69. The rank order of effects of canonical base pair substitutions at each position on kcat/Km was used to assess the involvement of major groove functional groups in recognition. Conclusions based on the biochemical data are largely consistent with the interactions revealed by the refined structure of the homologous Thermus thermophilus tRNA(Ser)-SerRS complex that Cusack and colleagues report in the accompanying paper. Disruption of an end-on hydrophobic interaction between the major groove C5(H) of pyrimidine 69 and an aromatic side chain of SerRS is shown to significantly decrease kcat/Km of a minihelix substrate. This type of interaction provides a means by which proteins can recognize the binary information of 'degenerate' sequences, such as the purine-pyrimidine base pairs of tRNA(Ser). The 3--70 base pair is shown to contribute to recognition by SerRS even though it is not contacted specifically by the protein. The latter effect derives from the organization of the specific contacts that SerRS makes with the neighboring 2--71 and 4--69 acceptor stem base pairs.

Evidence that specificity of microhelix charging by a class I tRNA synthetase occurs in the transition state of catalysis.

Determinants for the identities of tRNAs are located in the acceptor stem and, commonly, in the anticodon as well. Although the anticodon is an important determinant for the identity of methionine tRNA, RNA microhelices whose sequences are based on the acceptor stem alone can be aminoacylated by the class I methionyl-tRNA synthetase. We show here that specific nucleotide substitutions in a microhelix significantly reduced its rate of aminoacylation. In contrast, affinity coelectrophoresis analysis showed that microhelix binding to the enzyme was not significantly affected by the same substitutions. These and additional experiments and considerations imply that specific determinants for microhelix aminoacylation are needed for orientation of the acceptor stem in the transition state of catalysis rather than for enhanced binding interactions. The effect of linking together acceptor stem interactions with those in the anticodon, as occurs in the whole tRNA molecule, was also evaluated. This analysis showed that linkage results in some of the favorable acceptor stem and anticodon interactions being used to offset the free energy cost of straining the structure of the enzyme-tRNA complex.

Role of lysine-195 in the KMSKS sequence of E. coli tryptophanyl-tRNA synthetase

Lysine 195 in the K 195 MSKS sequence of E. coli tryptophanyl-tRNA synthetase (TrpRS) was replaced with alanine. The resulting K195A mutant TrpRS had essentially unchanged K m values for ATP and Trp, but a 1500-fold decreased k cat in a pyrophosphate-ATP exchange reaction. This large decrease in k cat reduces the rate of aminoacyladenylate formation (step 1) to a rate comparable to the rate of aminoacylation of tRNAT Trp (step 2) by the K195A mutant enzyme. Both the TIGN and KMSKS sequences are important for step 1 of class I aminoacyl-tRNA synthetase reactions.

Analysis of the isoleucyl-tRNA synthetase reaction by total rate equations. Magnesium and spermidine in the tRNA kinetics.

Derivation of a steady-state rate equation for the aminoacyl-tRNA synthetases is described, and its suitability for the analysis of various details of the reaction is tested. The equation is applied to the magnesium and spermidine dependences of the isoleucyl-tRNA synthetase reaction. Earlier work [Airas, R.K. (1990) Eur. J. Biochem. 192, 401-409] is expanded by experiments and calculations of the tRNA kinetics. The analysis suggests the following new details in addition to the earlier results: (a) The binding of tRNA to the enzyme (and not only the rate of the aminoacylation reaction) is affected by the presence of the Mg2+ and spermidine in the tRNA molecule. At least two bound Mg2+ or spermidines are required. (b) tRNA and PPi partly inhibit the binding of each other to the enzyme. (c) The transfer reaction is rather slow, and, at least under some conditions, it participates in rate limitation. (d) A Mg(2+)-induced reduction in the aminoacylation rate seems to be directed to the dissociation of the aminoacyl-tRNA from the enzyme. This dissociation rate is enhanced if a Mg2+ is first dissociated from the enzyme or tRNA. An increase in the Mg2+ concentration shifts the rate limitation from the transfer reaction towards dissociation of the product.

The rhythm of protein synthesis does not depend on oscillations of ATP level.

A rhythm of the [3H]leucine incorporation rate with a period of about one hour (circahoralian rhythm) has been found in rat hepatocytes grown in vitro as a monolayer and in liver organ culture. The periodicity of the incorporation rate remained after correction for changes in leucine pool size. A similar periodicity of the leucine incorporation rate was detected in a cell-free system prepared from rat hepatocytes. We have also found circahoralian oscillations of the ATP level and similar oscillations of the leucine tRNA aminoacylation rate in a hepatocyte monolayer. The addition of 1 mM ADP to the culture resulted in a considerable increase in the ATP level in the cells, but the rhythm of protein synthesis was retained under these conditions. The conclusion that there is a flexible association between changes in the ATP and GTP levels on the one hand, and oscillations of the protein synthesis rate on the other, is also supported by experiments with a cell-free system, in which the rhythm of protein synthesis rate was observed in the presence of excess ATP and GTP. We propose an hypothesis to explain the fractal pattern of circahoralian metabolic rhythms.

A comparative study of the relationship between thermostability and function of phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophilus

The relationship between thermostability and functional activities of phenylalanyl-tRNA synthetases (EC 6.1.1.20) from E. coli and Thermus thermophilus has been studied. In the case of the E. coli enzyme, the activity decreased after the 43°C treatment, both in the [ 32P]PP i-ATP exchange reaction and the overall aminoacylation reaction, due to thermo-inactivation of the phenylalanyl-tRNA synthetase, whereas tRNA Phe preserved its native structure. In the Th. thermophilus system, the enzyme showed extreme thermostability (up to 90°C), and the reduction in the tRNA aminoacylation rate after the 78°C treatment was ascribed to denaturation of the tRNA Phe. Since the enzyme did not lose the [ 32P]PP i-ATP exchange activity up to 80°C, the observed lower thermo-resistance of the tRNA is evidence that the native structure of ribonucleic acids should be one of the most difficult to stabilize at high temperatures.

Aminoacylation of 3′ terminal tRNA-like fragments of turnip yellow mosaic virus RNA: The influence of 5′ nonviral sequences

The present model of the L-shaped tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA encompasses 82 nucleotides. A previous kinetic study on 3′ terminal TYMV RNA fragments that contain the tRNA-like structure and a 5′ nonviral GGGAGA sequence, suggested that viral sequences upstream of the tRNA-like domain, i.e., upstream of nucleotide 82, increase the rate of aminoacylation (Dreher et al. (1988) Biochimie 70, 1719–1727). Here we report an increase in the aminoacylation rate when the number of nonviral nucleotides at the 5′ end of TYMV RNA transcripts was reduced. The influence of these 5′ proximal nonviral sequences on the conformation of the RNA molecule was investigated by structure mapping experiments. A structure that deviates from the tRNA-like structure was found in some of the transcripts. The formation of this alternative structure is dependent upon: (1) the nature and number of the nonviral nucleotides; (2) the number and secondary structure of viral nucleotides between the nonviral nucleotides and the tRNA-like domain. Footprinting experiments with valyl-tRNA synthetase from yeast suggest that the enzyme does not recognize the alternative structure.