Altered sperm tsRNAs in aged male contribute to anxiety‐like behavior in offspring

Parental age at first pregnancy is increasing worldwide. The offspring of aged father has been associated with higher risk of several neuropsychiatric disorders, such as schizophrenia and autism, but the underlying mechanism remains elusive. Here we report that advanced paternal age in mice alters the profile of transfer RNA‐derived small RNAs (tsRNAs). Injection of sperm tsRNAs from aged male mice into zygotes induced anxiety‐like behaviors in F1 males. RNA sequencing of the cerebral cortex and hippocampus of those F1 male mice altered the gene expression of dopaminergic synapse and neurotrophin. tsRNAs from aged male mice injection also altered the neuropsychiatry‐related gene expression in two‐cell and blastocyst stage embryos. More importantly, the sperm tsRNA profile changes significantly during aging in human. The up‐regulated sperm tsRNA target genes were involved in neurogenesis and nervous system development. These results suggest that aging‐related changes of sperm tsRNA may contribute to the intergenerational transmission of behavioral traits.     

Novel Temperature-Sensitive Mutants of Escherichia coli That Are Unable To Grow in the Absence of Wild-Type tRNA6Leu

Escherichia coli has only a single copy of a gene for tRNA₆^Leu (Y. Komine et al., J. Mol. Biol. 212:579–598, 1990). The anticodon of this tRNA is CAA (the wobble position C is modified to O²-methylcytidine), and it recognizes the codon UUG. Since UUG is also recognized by tRNA₄^Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O²-methyluridine) as its anticodon, tRNA₆^Leu is not essential for protein synthesis. The BT63 strain has a mutation in the anticodon of tRNA₆^Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su⁺6). We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA₆^Leu. These tRNA₆^Leu-requiring mutants were classified into two groups. The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions. Overexpression of the gene for tRNA₄^Leu restored the growth of group I mutants at 42°C. Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants. These results suggest that unmodified tRNA₄^Leu poorly recognizes the UUG codon at 42°C and that the wild-type tRNA₆^Leu is required for translation in order to maintain cell viability. The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division. The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants. The mechanism of requirement for tRNA₆^Leu remains to be investigated.In the universal genetic code, 61 sense codons correspond to 20 amino acids, and the various tRNA species mediate the flow of information from the genetic code to amino acid sequences. Since codon-anticodon interactions permit wobble pairing at the third position, 32 tRNAs, including tRNA^fMet, should theoretically be sufficient for a complete translation system. Although some organisms have fewer tRNAs (1), most have abundant tRNA species and multiple copies of major tRNAs. For example, Escherichia coli has 86 genes for tRNA (79 genes identified in reference 14, 6 new ones reported in reference 3, and one fMet tRNA at positions 2945406 to 2945482) that encode 46 different amino acid acceptor species. Although abundant genes for tRNAs are probably required for efficient translation, the significance of the apparently nonessential tRNAs has not been examined.E. coli has five isoaccepting species of tRNA^Leu. According to the wobble rule, tRNA₁^Leu recognizes only the CUG codon. The CUG codon is also recognized by tRNA₃^Leu (tRNA₂^Leu) and thus tRNA₁^Leu may not be essential for protein synthesis. Similarly, tRNA₆^Leu is supposed to recognize only the UUG codon, but tRNA₄^Leu can recognize both UUA and UUG codons. Thus, tRNA₆^Leu appears to be dispensable. The existence of an amber suppressor mutation of tRNA₆^Leu (supP, Su⁺6) supports this possibility. tRNA₆^Leu is encoded by a single-copy gene, leuX (supP), and Su⁺6 has a mutation in the anticodon, which suggests loss of the ability to recognize UUG (26). Why are so many species of tRNA^Leu required? Holmes et al. (12) examined the utilization of the isoaccepting species of tRNA^Leu in protein synthesis and showed that utilization differs depending on the growth medium; in minimal medium, isoacceptors tRNA₂^Leu (cited as tRNA₃^Leu; see Materials and Methods) and tRNA₄^Leu are the predominant species that are found bound to ribosomes, but an increased relative level of tRNA₁^Leu is found bound to ribosomes in rich medium. The existence of tRNA₆^Leu is puzzling. This isoaccepting tRNA accounts for approximately 10% of the tRNA^Leu in total-cell extracts. However, little if any tRNA₆^Leu is found on ribosomes in vivo, and it is also only weakly active in protein synthesis in vitro with mRNA from E. coli (12). It thus appears that tRNA₆^Leu is only minimally involved in protein synthesis in E. coli.To investigate the role of tRNA₆^Leu in E. coli, we attempted to isolate tRNA₆^Leu-requiring mutants from an Su⁺6 strain. These mutants required wild-type tRNA₆^Leu for survival at a nonpermissive temperature. We report here the isolation and the characterization of these mutants.     

Sporadic Distribution of tRNACCUArg Introns among α-Purple Bacteria: Evidence for Horizontal Transmission and Transposition of a Group I Intron

A group I intron interrupts the tRNA_CCU^Arg gene of the α-purple bacterium Agrobacterium tumefaciens (B. Reinhold-Hurek and D. A. Shub, Nature [London] 357:173–176, 1992). In this study, we assess the distribution of the corresponding intron among 12 additional species of α-purple bacteria. Of 10 newly identified tRNA_CCU^Arg genes, we found only two that contained an intron homologous to that of the Agrobacterium tRNA_CCU^Arg intron. This restricted and scattered distribution of the tRNA_CCU^Arg intron among α-purple bacteria is consistent with a recent origin and horizontal transmission. Primary and secondary structural similarities between tRNA_UAA^Leu introns found in strains of the cyanobacterium Microcystis aeruginosa (K. Rudi and K. S. Jacobsen, FEMS Microbiol. Lett. 156:293–298, 1997) and α-purple tRNA_CCU^Arg introns suggest that these introns share a more recent common ancestor than either does with other known cyanobacterial tRNA_UAA^Leu introns.     

Modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-tRNA synthetase

To prevent potential errors in protein synthesis, some aminoacyl-transfer RNA (tRNA) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Class Ia leucyl-tRNA synthetase (LeuRS) may misactivate various natural and non-protein amino acids and then mischarge tRNA^Leu. It is known that the fidelity of prokaryotic LeuRS depends on multiple editing pathways to clear the incorrect intermediates and products in the every step of aminoacylation reaction. Here, we obtained human cytoplasmic LeuRS (hcLeuRS) and tRNA^Leu (hctRNA^Leu) with high activity from Escherichia coli overproducing strains to study the synthetic and editing properties of the enzyme. We revealed that hcLeuRS could adjust its editing strategy against different non-cognate amino acids. HcLeuRS edits norvaline predominantly by post-transfer editing; however, it uses mainly pre-transfer editing to edit α-amino butyrate, although both amino acids can be charged to tRNA^Leu. Post-transfer editing as a final checkpoint of the reaction was very important to prevent mis-incorporation in vitro. These results provide insight into the modular editing pathways created to prevent genetic code ambiguity by evolution.     

Recognition of tRNALeu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps

Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNA^Leu recognition sets and their evolution, the recognition of tRNA^Leu by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8–A14, G18–U55 and G19–C56; and the orientation of the variable arm are critical elements for tRNA^Leu aminoacylation. Although dispensable for aminoacylation, the anticodon arm carries discrete editing determinants that are required for stabilizing the conformation of the post-transfer editing state and for promoting translocation of the tRNA acceptor arm from the synthetic to the editing site.     

The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase

Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3′ aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA^Leu (anticodon 5′-CAG-3′) isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA^Leu (CAG)—a G₃:C₇₀ base pair and a set of 4 nt (C₇₂, A₄:U₆₉, C₆₈)—that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.     

Export of Precursor tRNAIle from the Nucleus to the Cytoplasm in Human Cells

In the current concept, tRNA maturation in vertebrate cells, including splicing of introns, trimming of 5’ leader and 3’ trailer, and adding of CCA, is thought to occur exclusively in the nucleus. Here we provide evidence to challenge this concept. Unspliced intron-containing precursor tRNA^Ile was identified in Human Immunodeficiency Virus type 1 (HIV-1) virions, which are synthesized in the cytoplasm. Northern blot, confocal microscopy and quantitative RT-PCR further verified enrichment of this unspliced tRNA^Ile within the cytoplasm in human cells. In addition to containing an intron, the cytoplasmic precursor tRNA^Ile also contains a short incompletely processed 5´ leader and a 3´ trailer, which abundance is around 1000 fold higher than the nuclear precursor tRNA^Ile with long 5’ leader and long 3’ trailer. In vitro data also suggest that the cytoplasmic unspliced end-immature precursor tRNA^Ile could be processed by short isoform of RNase Z, but not long isoform of RNase Z. These data suggest that precursor tRNAs could export from the nucleus to the cytoplasm in human cells, instead of be processed only in the nucleus.     

Two tyrosine residues outside the editing active site in Giardia lamblia leucyl-tRNA synthetase are essential for the post-transfer editing

Leucyl-tRNA synthetase (LeuRS) is responsible for the Leu-tRNA^Leu synthesis. The connective peptide 1 (CP1) domain inserted into the Rossmann nucleotide binding fold possesses editing active site to hydrolyze the mischarged tRNA^Leu with noncognate amino acid, then to ensure high fidelity of protein synthesis. A few co-crystal structures of LeuRS with tRNA^Leu in different conformations revealed that tRNA^Leu 3′ end shuttled between synthetic and editing active sites dynamically with direct and specific interaction with the CP1 domain. Here, we reported that Y515 and Y520 outside the editing active site of CP1 domain of Giardia lamblia LeuRS (GlLeuRS) are crucial for post-transfer editing by influencing the binding affinity with mischarged tRNA^Leu. Mutations on Y515 and Y520 also decreased tRNA^Leu charging activity to various extents but had no effect on leucine activation. Our results gave some biochemical knowledge about interaction of tRNA^Leu 3′ end with the CP1 domain in archaeal/eukaryotic LeuRS.     

YibK is the 2′-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNALeu isoacceptors

Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few notable exceptions include the 2′-O-methyltransferase(s) that methylate the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNA^Leu_CmAA and tRNA^Leu_cmnm5UmAA. Here, we have used a comparative genomics approach to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 2′-O-methylation at position 34 in both tRNA^Leu_CmAA and tRNA^Leu_cmnm5UmAA. Loss of YibK methylation reduces the efficiency of codon–wobble base interaction, as demonstrated in an amber suppressor supP system. Inactivation of yibK had no detectable effect on steady-state growth rate, although a distinct disadvantage was noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase was impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK. Despite being one of the smallest characterized α/β knot proteins, YibK independently catalyzes the methyl transfer from S-adenosyl-L-methionine to the 2′-OH of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N⁶-(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL.     

Pigment Epithelium-derived Factor (PEDF) Prevents Retinal Cell Death via PEDF Receptor (PEDF-R): IDENTIFICATION OF A FUNCTIONAL LIGAND BINDING SITE*

The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu¹⁵⁹–Met³²⁵) with affinity similar to the full-length PEDF-R (Met¹–Leu⁵⁰⁴). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile¹⁹³–Leu²³²) and P1 (Thr²¹⁰–Leu²⁴⁹) peptides. Recombinant C-terminal truncated PEDF-R4 (Met¹–Leu²³²) and internally truncated PEDF-R and PEDF-R4 (ΔHis²⁰³–Leu²³²) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His²⁰³–Leu²³² region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.     

Excess of charged tRNALys maintains low levels of peptidyl-tRNA hydrolase in pth(Ts) mutants at a non-permissive temperature

Cellular changes have been monitored during the suppression, mediated by the overproduction of tRNA^Lys, of thermosensitivity in Escherichia coli strain AA7852 carrying a mutation in peptidyl-tRNA hydrolase (Pth) encoded by the pth(Ts) gene. The presence in AA7852 cells of a plasmid bearing lysV gene helped to maintain low levels of the unstable Pth(Ts) protein and to preserve the viability of the mutant line at 41°C whereas plasmids bearing other tRNA genes were ineffective. At 32°C the excess of tRNA^Lys did not alter the percentages of the free-, charged- or peptidyl-tRNA^Lys species compared with those found in strains that did not overproduce tRNA^Lys. At 41°C, however, despite increases in the level of peptidyl-tRNA^Lys, the excess tRNA^Lys helped to maintain the concentration of charged-tRNA^Lys at a level comparable with that found in non-overproducer cells grown at a permissive temperature. In addition, the excess tRNA^Lys at 41°C provoked a reduction in the concentrations of various peptidyl-tRNAs, which normally accumulate in pth(Ts) cells, and a proportional increase in the concentrations of the corresponding aminoacyl-tRNAs. The possible mechanism of rescue due to the overexpression of tRNA^Lys and the causes of tRNA^Lys starvation in pth(Ts) strains grown at non-permissive temperatures are considered.     

Localization of four genes for three tRNALeu's on the physical map of the soybean (Glycine max L.) chloroplast genome

Three tRNAs^Leu from soybean chloroplasts were isolated and hybridized to restriction fragments of soybean chloroplast DNA. Based on the hybridization pattern, the locations of four genes coding for tRNA₁^Ley, tRNA₂^Leu (two genes tRNA_2a^Ley and tRNA_2b^Leu, are present in the inverted repeat region) and tRNA₃^Leu were determined on the physical map of the soybean chloroplast genome.     

PANDORA-Seq unveils the hidden small noncoding RNA landscape in atherosclerosis of LDL receptor-deficient mice

Small noncoding RNAs (sncRNAs) play diverse roles in numerous biological processes. While the widely used RNA sequencing (RNA-Seq) method has advanced sncRNA discovery, RNA modifications can interfere with the complementary DNA library construction process, preventing the discovery of highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that may have important functions in disease development. To address this technical obstacle, we recently developed a novel PANDORA-Seq (Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing) method to overcome RNA modification-elicited sequence interferences. To identify novel sncRNAs associated with atherosclerosis development, LDL receptor-deficient (LDLR^−/−) mice were fed a low-cholesterol diet or high-cholesterol diet (HCD) for 9 weeks. Total RNAs isolated from the intima were subjected to PANDORA-Seq and traditional RNA-Seq. By overcoming RNA modification-elicited limitations, PANDORA-Seq unveiled an rsRNA/tsRNA-enriched sncRNA landscape in the atherosclerotic intima of LDLR^−/− mice, which was strikingly different from that detected by traditional RNA-Seq. While microRNAs were the dominant sncRNAs detected by traditional RNA-Seq, PANDORA-Seq substantially increased the reads of rsRNAs and tsRNAs. PANDORA-Seq also detected 1,383 differentially expressed sncRNAs induced by HCD feeding, including 1,160 rsRNAs and 195 tsRNAs. One of HCD-induced intimal tsRNAs, tsRNA-Arg-CCG, may contribute to atherosclerosis development by regulating the proatherogenic gene expression in endothelial cells. Overall, PANDORA-Seq revealed a hidden rsRNA and tsRNA population associated with atherosclerosis development. These understudied tsRNAs and rsRNAs, which are much more abundant than microRNAs in the atherosclerotic intima of LDLR^−/− mice, warrant further investigations.     

Modulation of Aminoacylation and Editing Properties of Leucyl-tRNA Synthetase by a Conserved Structural Module

A conserved structural module following the KMSKS catalytic loop exhibits α-α-β-α topology in class Ia and Ib aminoacyl-tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl-tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third α-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNA^Leu, which decreases both aminoacylation and editing activities. Two glycine residues on this α-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the β-strand enhance the editing activity of EcLeuRS and sense the size of the tRNA^Leu D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS.     

Temperature dependent mistranslation in a hyperthermophile adapts proteins to lower temperatures

All organisms universally encode, synthesize and utilize proteins that function optimally within a subset of growth conditions. While healthy cells are thought to maintain high translational fidelity within their natural habitats, natural environments can easily fluctuate outside the optimal functional range of genetically encoded proteins. The hyperthermophilic archaeon Aeropyrum pernix (A. pernix) can grow throughout temperature variations ranging from 70 to 100°C, although the specific factors facilitating such adaptability are unknown. Here, we show that A. pernix undergoes constitutive leucine to methionine mistranslation at low growth temperatures. Low-temperature mistranslation is facilitated by the misacylation of tRNA^Leu with methionine by the methionyl-tRNA synthetase (MetRS). At low growth temperatures, the A. pernix MetRS undergoes a temperature dependent shift in tRNA charging fidelity, allowing the enzyme to conditionally charge tRNA^Leu with methionine. We demonstrate enhanced low-temperature activity for A. pernix citrate synthase that is synthesized during leucine to methionine mistranslation at low-temperature growth compared to its high-fidelity counterpart synthesized at high-temperature. Our results show that conditional leucine to methionine mistranslation can make protein adjustments capable of improving the low-temperature activity of hyperthermophilic proteins, likely by facilitating the increasing flexibility required for greater protein function at lower physiological temperatures.     

Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding

Transfer RNA structure involves complex folding interactions of the TΨC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TΨC domain, rT₅₄, Ψ₅₅ and m⁵C₄₉, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA^Phe T-half molecules (nucleosides 40–72) with the corresponding unmodified D-half molecule (nucleosides 1–30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg²⁺ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K_ds (rT₅₄ = 6 ± 2, m⁵C₄₉ = 11 ± 2, Ψ₅₅ = 14 ± 5, and rT₅₄,Ψ₅₅ = 11 ± 3 µM) significantly lower than that of the unmodified T-half (40 ± 10 µM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA^Phe and native yeast tRNA^Phe, as determined by lead cleavage patterns at U₁₇ and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA’s TΨC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.     

In vivo identification of essential nucleotides in tRNALeu to its functions by using a constructed yeast tRNALeu knockout strain

The fidelity of protein biosynthesis requires the aminoacylation of tRNA with its cognate amino acid catalyzed by aminoacyl-tRNA synthetase with high levels of accuracy and efficiency. Crucial bases in tRNA^Leu to aminoacylation or editing functions of leucyl-tRNA synthetase have been extensively studied mainly by in vitro methods. In the present study, we constructed two Saccharomyces cerevisiae tRNA^Leu knockout strains carrying deletions of the genes for tRNA^Leu(GAG) and tRNA^Leu(UAG). Disrupting the single gene encoding tRNA^Leu(GAG) had no phenotypic consequence when compared to the wild-type strain. While disrupting the three genes for tRNA^Leu(UAG) had a lethal effect on the yeast strain, indicating that tRNA^Leu(UAG) decoding capacity could not be compensated by another tRNA^Leu isoacceptor. Using the triple tRNA knockout strain and a randomly mutated library of tRNA^Leu(UAG), a selection to identify critical tRNA^Leu elements was performed. In this way, mutations inducing in vivo decreases of tRNA levels or aminoacylation or editing ability by leucyl-tRNA synthetase were identified. Overall, the data showed that the triple tRNA knockout strain is a suitable tool for in vivo studies and identification of essential nucleotides of the tRNA.