Dihydrouridine Loop Research Articles

Complete mitogenomes of Turkish tree squirrels, Sciurus anomalus and S. vulgaris, (Sciuridae: Rodentia: Mammalia) and their phylogenetic status within the tribe Sciurini

The genus Sciurus, a member of the family Sciuridae, is widely distributed in the Holarctic region. To better understand mitogenomic characteristics and to reveal internal phylogenetic relationships of the genus, 20 complete mitogenomes of Turkish tree squirrels were successfully sequenced for the first time, including 19 for S. anomalus (from 16,505 bp to 16,510 bp) and one for S. vulgaris (16,511 bp). The mitogenomes of two species were AT-biased. All tRNAs for two species displayed a typical clover-leaf structure, except for tRNASer(AGY). The tRNA Serine1 (S1)-GCT structure lacked the dihydrouridine (DHU) loop and stem. Based on mitogenomic dataset for phylogeny of Sciurinae, phylogenetic analyses (Bayesian Inference and Maximum Likelihood) did not support monophyly of Sciurus and proposed that S. anomalus, the most basal taxa in the Sciurini tribe, had at least five mitogenome lineages, which were also supported by network analysis. The dissimilarities among the five lineages of S. anomalus ranged from 0.0042 (0.42%) to 0.0062 (0.62%) using K2P sequence pairwise distances. In addition to this mitogenomic analysis result, phylogenetic analyses using the CYTB + D-loop dataset proposed the existence of at least nine lineages for S. anomalus, which was different than those of the previous studies. The current study proposed that the use of mitogenomic data for reconstructing the phylogeny of Turkey’s Sciurus holds an important value for revealing evolutionary relationships.

Structure and comparative analysis of the mitochondrial genomes of Liolaemus lizards with different modes of reproduction and ploidy levels.

Liolaemus is the most specious genus of the Squamata lizards in South America, presenting exceptional evolutionary radiation and speciation patterns. This recent diversification complicates the formal taxonomic treatment and the phylogenetic analyses of this group, causing relationships among species to remain controversial. Here we used Next-Generation Sequencing to do a comparative analysis of the structure and organization of the complete mitochondrial genomes of three differently related species of Liolaemus and with different reproductive strategies and ploidy levels. The annotated mitochondrial genomes of ca. 17 kb are the first for the Liolaemidae family. Despite the high levels of sequence similarity among the three mitochondrial genomes over most of their lengths, the comparative analyses revealed variations at the stop codons of the protein coding genes and the structure of the tRNAs among species. The presence of a non-canonical dihydrouridine loop is a novelty for the pleurodonts iguanians. But the highest level of variability was observed in two repetitive sequences of the control region, which were responsible for most of the length heterogeneity of the mitochondrial genomes. These tandem repeats may be useful markers to analyze relationships of closely related species of Liolaemus and related genera and to conduct population and phylogenetic studies.

Overexpression, Purification, and Preliminary Crystallographic Analysis of the Archaeosine Synthase GAT‐QueC

Archaeosine (G+) is a highly modified nucleosides found exclusively at position‐15 in the dihydrouridine loops (D‐loop) of archaeal tRNAs where it contributes to tRNA structural stability. In a late step in G+ biosynthesis, the precursor nucleoside 7‐cyano‐7‐deazaguanine (preQ0) is inserted into tRNA by the enzyme tRNA‐guanine transglycosylase (arcTGT) before conversion to G+ by archaeosine synthases that vary among archaeal phyla: ArcS in Euryarchaeota, and QueF‐like or GAT‐QueC in Chrenoarchaeota. GAT‐QueC is a two‐domain enzyme harboring an N‐terminal glutamine amidotransferase (GAT) domain, and a C‐terminal domain homologous to QueC, the enzyme that produces preQ0. Consistent with its 2‐domain structure, GAT‐QueC functions in vivo as both a preQ0 synthase and an amidinotransferase, generating G+‐modified tRNA. However, it remains unknown whether the GAT domain acts on preQ0 before or after its insertion into tRNA by arcTGT. Here we describe overexpression, purification and preliminary biochemical and crystallographic characterization of GAT‐QueC from Sulfolobus tokodaii (StGAT‐QueC). Analytical gel filtration chromatography and electrophoretic mobility shift analysis reveal that StGAT‐QueC is a monomeric protein with molecular weight of 55 kDa and no affinity for tRNA, suggesting that the enzyme acts on free preQ0, not preQ0‐modified tRNA. High throughput crystallization of the apoenzyme (and in the presence of preQ0) using the vapor diffusion method yielded crystals that grew in 21 days from solutions containing methyl pentanediol and sodium acetate, and diffracted to 5.8 Å without cryo protection. The crystals belong to cubic space group F23, exhibit unit cell length=292 Å and contain 4 protein molecules per asymmetric unit.Support or Funding InformationNIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).

Archaeosine Modification of Archaeal tRNA: Role in Structural Stabilization.

Archaeosine (G+) is a structurally complex modified nucleoside found quasi-universally in the tRNA of Archaea and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside the Archaea G+ is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G+ at position 15, coupled with its novel molecular structure, led to a hypothesis that G+ stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G+ contributes to tRNA stability and define the biological role of G+, we investigated the consequences of introducing targeted mutations that disrupt the biosynthesis of G+ into the genome of the hyperthermophilic archaeon Thermococcus kodakarensis and the mesophilic archaeon Methanosarcina mazei, resulting in modification of the tRNA with the G+ precursor 7-cyano-7-deazaguansine (preQ0) (deletion of arcS) or no modification at position 15 (deletion of tgtA). Assays of tRNA stability from in vitro-prepared and enzymatically modified tRNA transcripts, as well as tRNA isolated from the T. kodakarensis mutant strains, demonstrate that G+ at position 15 imparts stability to tRNAs that varies depending on the overall modification state of the tRNA and the concentration of magnesium chloride and that when absent results in profound deficiencies in the thermophily of T. kodakarensisIMPORTANCE Archaeosine is ubiquitous in archaeal tRNA, where it is located at position 15. Based on its molecular structure, it was proposed to stabilize tRNA, and we show that loss of archaeosine in Thermococcus kodakarensis results in a strong temperature-sensitive phenotype, while there is no detectable phenotype when it is lost in Methanosarcina mazei Measurements of tRNA stability show that archaeosine stabilizes the tRNA structure but that this effect is much greater when it is present in otherwise unmodified tRNA transcripts than in the context of fully modified tRNA, suggesting that it may be especially important during the early stages of tRNA processing and maturation in thermophiles. Our results demonstrate how small changes in the stability of structural RNAs can be manifested in significant biological-fitness changes.

The mitochondrial tRNAAla 5587T>C and tRNALeu(CUN) 12280A>G mutations may be associated with hypertension in a Chinese family.

Hypertension is a very common cardiovascular disorder, however, the molecular mechanism underlying this disease remains poorly understood. Recently, an increasing number of studies have demonstrated that mitochondrial (mt)DNA mutations serve important roles in the pathogenesis of hypertension. The current study reported the clinical and molecular characterization of a Chinese family with maternally inherited hypertension (the penetrance of hypertension was 71.4%). In addition, the entire mitochondrial transfer (mt-t)RNA genomes was amplified using a polymerase chain reaction (PCR) and identified through direct Sanger sequencing. Additionally, the mtDNA copy number in matrilineal relatives in this family was evaluated using quantitative PCR. The sequence analysis of the 22 mt-tRNA genes led to the identification of tRNAAla 5587T>C (thymine to cytosine) and tRNALeu(CUN) 12280A>G (adenine to guanine) mutations. Notably, the heteroplasmic 5587T>C mutation was located at the 3' end of tRNAAla (position 73), which is highly conserved from bacteria to human mitochondria. In addition, the 12280A>G mutation was revealed to occurs at the dihydrouridine loop of tRNALeu(CUN) (position 15) and may decrease the steady-state level of mt-tRNA. As a result, 5587T>C and 12280A>G mutations may lead to the failure of tRNAs metabolism and subsequently cause mitochondrial protein synthesis defects. Molecular analysis revealed that patients carrying the 5587T>C and 12280A>G mutations had a lower copy number of mtDNA compared with a control with hypertension, but without the mutations, suggesting that these mutations may cause mitochondrial dysfunctions that are responsible for hypertension. Therefore, mt-tRNAAla 5587T>C and tRNALeu(CUN) 12280A>G mutations may be involved in the pathogenesis of hypertension in this family.

Crystal Structure of the Archaeosine Synthase QueF‐Like – Insights into Amidino Transfer and tRNA Recognition by the Tunnel Fold

Crystal Structure of the Archaeosine Synthase QueF‐Like – Insights into Amidino Transfer and tRNA Recognition by the Tunnel Fold

MELAS: Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-Like Episodes

Mitochondrial disease is caused by a deficiency in the energy supply to cells due to mitochondrial dysfunction. Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is a mitochondrial disease that presents with stroke-like episodes such as acute onset of neurological deficits and characteristic imaging findings. Stroke-like episodes in MELAS have the following features: 1) neurological deficits due to localization of lesions in the brain, 2) episodes often accompany epilepsy, 3) lesions do not follow the vascular supply area, 4) lesions are more often seen in the posterior brain than in the anterior brain, 5) lesions spread to an adjacent area in the brain, and 6) neurological symptoms often disappear together with imaging findings, but later relapse. About 80% of patients with MELAS have an A-to-G transition mutation at the nucleotide pair 3243 in the dihydrouridine loop of mitochondrial tRNA^Leu(UUR), which causes the absence of posttranscriptional taurine modification at the wobble nucleotide of mitochondrial tRNA^Leu(UUR) and disrupts protein synthesis. However, the precise pathophysiology of stroke-like episodes is under investigation, with possible hypotheses for these episodes including mitochondrial angiopathy, mitochondrial cytopathy, and neuron-astrocyte uncoupling. With regard to treatment, L-arginine and taurine have recently been suggested for relief of clinical symptoms.

Length variation in the mouse mitochondrial tRNAArg DHU loop size promotes oxidative phosphorylation functional differences

The efficiency of the cellular oxidative phosphorylation system was recently shown to be modulated by common mitochondrial tRNA(A) (rg) haplotypes. The molecular mechanism by which some mt-Tr haplotypes induce these functional differences remains undetermined. Common polymorphisms in mouse mt-Tr genes affect the size of the dihydrouridine loop in the mature tRNA, producing loops of between five and seven nucleotides, the largest being a rare variant among mammals. Here, we analyzed a new mt-Tr variant identified in C3H mice, and found that it is mitochondrial tRNA loop size, but not the specific sequence, that is responsible for the observed differences in cellular respiration. We further found that the sensitivity of mitochondrial protein synthesis to specific inhibitors is dependent on the mt-Tr gene haplotype, and confirmed that the differences in oxidative phosphorylation performance are masked by a reactive oxygen species-induced compensatory increase in mitochondrial biogenesis.

Discovery and Characterization of an Amidinotransferase Involved in the Modification of Archaeal tRNA

The presence of the 7-deazaguanosine derivative archaeosine (G(+)) at position 15 in tRNA is one of the diagnostic molecular characteristics of the Archaea. The biosynthesis of this modified nucleoside is especially complex, involving the initial production of 7-cyano-7-deazaguanine (preQ(0)), an advanced precursor that is produced in a tRNA-independent portion of the biosynthesis, followed by its insertion into the tRNA by the enzyme tRNA-guanine transglycosylase (arcTGT), which replaces the target guanine base yielding preQ(0)-tRNA. The enzymes responsible for the biosynthesis of preQ(0) were recently identified, but the enzyme(s) catalyzing the conversion of preQ(0)-tRNA to G(+)-tRNA have remained elusive. Using a comparative genomics approach, we identified a protein family implicated in the late stages of archaeosine biosynthesis. Notably, this family is a paralog of arcTGT and is generally annotated as TgtA2. Structure-based alignments comparing arcTGT and TgtA2 reveal that TgtA2 lacks key arcTGT catalytic residues and contains an additional module. We constructed a Haloferax volcanii DeltatgtA2 derivative and demonstrated that tRNA from this strain lacks G(+) and instead accumulates preQ(0). We also cloned the corresponding gene from Methanocaldococcus jannaschii (mj1022) and characterized the purified recombinant enzyme. Recombinant MjTgtA2 was shown to convert preQ(0)-tRNA to G(+)-tRNA using several nitrogen sources and to do so in an ATP-independent process. This is the only example of the conversion of a nitrile to a formamidine known in biology and represents a new class of amidinotransferase chemistry.

The m.3244G>A mutation in mtDNA is another cause of progressive external ophthalmoplegia

We sequenced all mitochondrial tRNA genes in a 61-year-old man with chronic progressive external ophthalmoplegia and mitochondrial myopathy but without mtDNA rearrangements, and identified a heteroplasmic m.3244G>A mutation in the tRNA Leu(UUR) gene. This mutation had been previously associated with the MELAS phenotype, but not described in any detail. The mutation load in muscle was 84% and COX-negative fibers harbored greater levels of mutant genomes than COX-positive fibers. The m.3244G>A mutation affects a highly conserved nucleotide in the dihydrouridine loop and has been associated with a wobble modification deficiency of the mutant tRNA.

Core flexibility of a truncated metazoan mitochondrial tRNA

Secondary and tertiary structures of tRNAs are remarkably preserved from bacteria to humans, the notable exception being the mitochondrial (m) tRNAs of metazoans, which often deviate substantially from the canonical cloverleaf (secondary) or ‘L’-shaped (tertiary) structure. Many metazoan mtRNAs lack either the TψC (T) or dihydrouridine (D) loops of the canonical cloverleaf, which are known to confer structural rigidity to the folded structure. Thus, the absence of canonical TψC–D interactions likely results in greater dispersion of anticodon-acceptor interstem angle than for canonical tRNAs. To test this hypothesis, we have assessed the dispersion of the anticodon-acceptor angle for bovine mtRNASer(AGY), which lacks the canonical D arm and is thus incapable of forming stabilizing interarm interactions. Using the method of transient electric birefringence (TEB), and by changing the helical torsion angle between a core mtRNA bend and a second bend of known angle/rigidity, we have demonstrated that the core of mtRNASer(AGY) has substantially greater flexibility than its well-characterized canonical counterpart, yeast cytoplasmic tRNAPhe. These results suggest that increased flexibility, in addition to a more open interstem angle, would allow both noncanonical and canonical mtRNAs to utilize the same protein synthetic apparatus.

The Mitochondrial Myopathy Encephalopathy, Lactic Acidosis with??Stroke-Like Episodes (MELAS) Syndrome

Mitochondrial encephalomyopathies are a multisystemic group of disorders that are characterised by a wide range of biochemical and genetic mitochondrial defects and variable modes of inheritance. Among this group of disorders, the mitochondrial myopathy, encephalopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome is one of the most frequently occurring, maternally inherited mitochondrial disorders. As the name implies, stroke-like episodes are the defining feature of the MELAS syndrome, often occurring before the age of 15 years. The clinical course of this disorder is highly variable, ranging from asymptomatic, with normal early development, to progressive muscle weakness, lactic acidosis, cognitive dysfunction, seizures, stroke-like episodes, encephalopathy and premature death. This syndrome is associated with a number of point mutations in the mitochondrial DNA, with over 80% of the mutations occurring in the dihydrouridine loop of the mitochondrial transfer RNA(Leu(UUR)) [tRNA(Leu)((UUR))] gene. The pathophysiology of the disease is not completely understood; however, several different mechanisms are proposed to contribute to this disease. These include decreased aminoacylation of mitochondrial tRNA, resulting in decreased mitochondrial protein synthesis; changes in calcium homeostasis; and alterations in nitric oxide metabolism. Currently, no consensus criteria exist for treating the MELAS syndrome or mitochondrial dysfunction in other diseases. Many of the therapeutic strategies used have been adopted as the result of isolated case reports or limited clinical studies that have included a heterogeneous population of patients with the MELAS syndrome, other defects in oxidative phosphorylation or lactic acidosis due to disorders of pyruvate metabolism. Current approaches to the treatment of the MELAS syndrome are based on the use of antioxidants, respiratory chain substrates and cofactors in the form of vitamins; however, no consistent benefits have been observed with these treatments.

Aminoacylation of an unusual tRNA(Cys) from an extreme halophile.

The extreme halophile Halobacterium species NRC-1 overcomes external near-saturating salt concentrations by accumulating intracellular salts comparable to those of the medium. This raises the fundamental question of how halophiles can maintain the specificity of protein-nucleic acid interactions that are particularly sensitive to high salts in mesophiles. Here we address the specificity of the essential aminoacylation reaction of the halophile, by focusing on molecular recognition of tRNA(Cys) by the cognate cysteinyl-tRNA synthetase. Despite the high salt environments of the aminoacylation reaction, and despite an unusual structure of the tRNA with an exceptionally large dihydrouridine loop, we show that aminoacylation of the tRNA proceeds with a catalytic efficiency similar to that of its mesophilic counterparts. This is manifested by an essentially identical K(m) for tRNA to those of the mesophiles, and by recognition of the same nucleotide determinants that are conserved in evolution. Interestingly, aminoacylation of the halophile tRNA(Cys) is more closely related to that of bacteria than eukarya by placing a strong emphasis on features of the tRNA tertiary core. This suggests an adaptation to the highly negatively charged tRNA sugar-phosphate backbone groups that are the key elements of the tertiary core.

Interaction of HIV Tat Peptides With tRNAPhe from Yeast

We present data on the interaction of arginine-rich peptides of human immunodeficiency virus (HIV-Tat) with tRNAPhe of Saccharomyces cerevisiae. We have found that tRNA forms complexes with the Tat1 peptide of amino acid sequence GRKKRRQRRRA and its mutants where R is replaced by D-arginine, citrulline or ornithine. The structure of tRNA-Tat1 complex was probed by specific RNases digestions and Pb2+-induced cleavage of phosphodiester bond of guanosine. The nucleotide sequence UGGG located in the dihydrouridine loop of tRNAPhe binds to Tat peptide and therefore is specifically protected against RNases and is not hydrolyzed by Pb2+ ion. It seems that the peptide-RNA complex formation depends on direct recognition of guanine moieties of tRNA with arginine residues. These interactions are similar to those observed in many DNA-protein complexes, but are different from those previously observed for TAR RNA-Tat complexes.

Interaction of HIV Tat model peptides with tRNA and 5S rRNA.

New data are presented on the interaction of model synthetic peptides containing an arginine-rich region of human immunodeficiency virus (HIV-Tat), with native RNA molecules: tRNA(Phe) of Saccharomyces cerevisiae and 5S rRNA from Lupinus luteus. Both RNA species form complexes with the Tat1 (GRKKRRQRRRA) and Tat2 (GRKKRRQRRRAPQDSQTHQASLSKQPA) peptides, as shown by electrophoretic gel shift and RNase footprint assays, and CD measurements. The nucleotide sequence UGGG located in the dihydrouridine loop of tRNAPhe as well as in the loop D of 5S rRNA is specifically protected against RNases. Our data indicate direct interactions of guanine of RNA moieties with arginine residues. These interactions seem similar to those observed in DNA-protein complexes, but different from those previously observed in the TAR RNA-Tat complexes.

Mitochondrial Encephalomyopathy Associated with a Novel Mutation in the Mitochondrial tRNAleu(UUR)Gene (A3243T)

We report a new mutation, an A→T transition at nt 3243 in the mitochondrial tRNAleu(UUR)gene, in a 9-year-old girl who presented with muscle weakness of 3 years duration complicated by rapidly progressive encephalopathy. In muscle, the activity of the mitochondrial respiratory chain complexes I, III, and IV was markedly reduced. The mutation, involving a highly conserved base pair in the dihydrouridine loop, was heteroplasmic in muscle (81.4%), skin (69.3%), and blood (13.8%) and was not present in blood of 50 healthy individuals. The mitochondrial 3243 base is a “hot spot” for mutations; an A→G transition at this position is found in a high proportion in most MELAS patients. Since the A→T transtion creates a new recognition site for the restriction enzyme TspRI, both ApaI and TspRI should be used to exclude a mutation at nt 3243.

Structural analysis of the interaction of the tRNA modifying enzymes Tgt and QueA with a substrate tRNA

The enzymes tRNA guanine-transglycosylase (Tgt) and S-adenosylmethionine : tRNA ribosyltransferase-isomerase (QueA) participate in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q) in Escherichia coli. Here we show by HPLC analysis and gel retardation that both enzymes interact with an in vitro transcribed tRNA Asp from yeast, specifically modified with a Q precursor molecule. RNase I footprinting experiments showed strong protein tRNA contacts in the anticodon stem-loop and a minor interaction with the dihydrouridine loop. This suggests that all identity elements for the recognition of Q-specific tRNAs are clustered in the anticodon region and explains earlier results that both enzymes accept a RNA microhelix with the sequence of an anticodon stem-loop as substrate.

Permutation of a pair of tertiary nucleotides in a transfer RNA.

The tertiary nucleotides at positions 15 and 48 in a tRNA establish non-Watson-Crick hydrogen interactions that connect the dihydrouridine (D) loop with the variable loop and stabilize the "L"-shaped tRNA structure. Although the majority of tRNAs have G15.C48 or A15.U48, all of the 16 possible nucleotide pairs at positions 15 and 48 can be found in the existing cytoplasmic and mitochondrial tRNA sequences. Because tRNAs contain a variety of slightly different sets of tertiary nucleotides, this complexity raises the question of whether a given tRNA sequence framework can accommodate all of the 16 compositions at positions 15 and 48. In this work, G15 and C48 in an Escherichia coli alanine amber suppressor tRNA were permuted, and variants were tested for biological activity in vivo. All but an A15.A48 variant were functional, indicating substantial flexibility at positions 15 and 48 to accommodate nucleotide variations. Analysis of the A15.A48 variant with chemical probes showed that this mutant harbors a defect that specifically changes the conformation of the anticodon sequence. Interestingly, human tRNA(Ala) has A15.A48. Additional nucleotide substitutions in E. coli A15.A48 tRNA(Ala) that recreate the D loop sequence of human tRNA(Ala) restored the biological activity to this tRNA by reestablishing the wild-type conformation of the anticodon sequence. The results suggest a distal relationship between the D and the anticodon loops in a tRNA and delineate covariation of specific nucleotides in the evolution of tRNA(Ala) from E. coli to human.

Structural elements that contribute to an unusual tertiary interaction in a transfer RNA.

Transfer RNAs (tRNAs) contain a set of defined tertiary hydrogen-bonding interactions that are established between conserved and semiconserved nucleotides. Although the crystal structures of tRNAs describe each of the tertiary interactions in detailed molecular terms, little is known about the underlying structural parameters that stabilize the tertiary interactions. Escherichia coli (E. coli) tRNA(Cys) has an unusual tertiary interaction between G15 in the dihydrouridine (D) loop and G48 in the variable loop that is critical for cysteine aminoacylation. All other tRNAs have a purine 15 and a complementary pyrimidine 48 that establish a tertiary interaction known as the Levitt base pair [Levitt, M. (1969) Nature 224, 759-763; Klug et al. (1974) J. Mol. Biol. 89, 511-516]. In this study, the G15.G48 tertiary interaction in E. coli tRNA(Cys) was used to investigate the structural elements that contribute to its variation from the Levitt base pair. Analysis with chemical probes showed that substitution of U21 with A21 in the D loop and formation of a Watson-Crick base pair between nucleotides 13 and 22 in the D stem switch the hydrogen-pairing of G15.G48 to a Levitt-like G15.G48 base pair. This switch was accompanied by a decrease of the catalytic efficiency of aminoacylation by 2 orders of magnitude. In contrast, insertion of additional nucleotides in the D or variable loops had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Triple aminoacylation specificity of a chimerized transfer RNA.

We report here the rational design and construction of a chimerized transfer RNA with tripartite aminoacylation specificity. A yeast aspartic acid specific tRNA was transformed into a highly efficient acceptor of alanine and phenylalanine and a moderate acceptor of valine. The transformation was guided by available knowledge of the requirements for aminoacylation by each of the three amino acids and was achieved by iterative changes in the local sequence context and the structural framework of the variable loop and the two variable regions of the dihydrouridine loop. The changes introduced to confer efficient acceptance of the three amino acids eliminate aminoacylation with aspartate. The interplay of determinants and antideterminants for different specific aminoacylations, and the constraints imposed by the structural framework, suggest that a tRNA with an appreciable capacity for more than three efficient aminoacylations may be inherently difficult to achieve.