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We have used site-specific mutagenesis to change the anticodon of a Xenopus laevis tyrosine tRNA gene so that it would recognize ochre codons. This tRNA gene is expressed when amplified in monkey cells as part of a SV40 recombinant and efficiently suppresses termination at both the ochre codon separating the adenovirus 2 hexon gene from a 23-kd downstream gene and the ochre codon at the end of the NS1 gene of influenza virus A/Tex/1/68. Termination at an amber codon of a NS1 gene of another influenza virus strain was not suppressed by the (Su+) ochre gene suggesting that in mammalian cells amber codons are not recognized by ochre suppressor tRNAs. Finally, microinjection into mammalian cells of both (Su+) ochre tRNA genes and selectible genes containing ochre nonsense mutations gives rise to colonies under selective conditions. We conclude that it should be possible to isolate a wide assortment of mammalian cell lines with ochre suppressor activity.

The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.

The nucleotide sequences at the 5' termini of rabbit alpha and beta globin mRNAs have been determined. Periodate oxidation of globin mRNA followed by reduction with 3H-sodium borohydride and subsequent analysis of the 3H-labeled mRNA reveals the presence of the "cap" structure m7G5'ppp- blocking the 5' terminus. After periodate oxidation, beta elimination, and phosphomonoesterase treatment to remove the m7G5'ppp- "cap," the 5' end of globin mRNA was labeled with 32P using gamma-32P-ATP and T4 polynucleotide kinase. The 5'-32P-labeled alpha and beta globin mRNAs were then resolved from each other by polyacrylamide gel electrophoresis under denaturing conditions and sequenced separately. The 5' terminal nucleotide sequences determined are: alpha--m7G5'ppp5m6AmC(m)ACUUCUGG- BETA--M7G5'ppp5m6AmC(m) ACUUGCUUUUGACACAA Besides the m7G "cap" structure, the two sequences are identical for the first six nucleotides and then diverge. No initiator codon is present within the first ten nucleotides from the 5' end of the alpha globin mRNA, and the first nineteen nucleotides from the 5' end of beta globin mRNA.

We have examined transcripts corresponding to the Halobacterium halobium bacterio-opsin (BO) gene in wild-type and in BO-deficient mutant strains containing the insertion elements ISH1 or ISH2 in the BO gene. BO mRNA from the wild-type strain was purified by hybrid selection using single-stranded cDNA. Labeling by vaccinia virus capping enzyme and [alpha-(32)P]GTP showed that it contains the 5'-terminal nucleotide of the primary transcript. Sequence analysis showed that transcription begins only two nucleotides upstream of the initiator codon for BO. Two species of BO mRNA were found; the major species has a ragged 3' terminus approximately 45 nucleotides downstream from the terminator codon for BO, while the minor species is about 170 nucleotides longer at the 3' end. Analysis of the transcripts in several BO gene mutant strains by RNA gel-transfer hybridization showed that (i) mutants with ISH1 insertions within the NH(2)-terminal coding region of the gene contain no detectable transcripts, (ii) mutants with ISH2 near the middle of the coding region of the gene contain multiple incomplete transcripts, and (iii) a mutant that is partially BO deficient due to an insertion of ISH2 100 base pairs upstream of the site of initiation of transcription contains a decreased level of BO mRNA.

The 5S RNA genes (5S genes) in N. crassa are not tandemly arranged or tightly clustered as in other eucaryotes that have been examined. 55 RNA or cloned 5S DNA hybridizes to at least 30 different restriction fragments of Neurospora DNA. Of 34 5S DNA clones examined, each contains a single 5S gene. Saturation hybridization analyses indicate that there are about 100 copies of 5S genes in the genome of this organism. We have partially or completely sequenced the 5S region of 15 clones. Both identical and highly divergent 5S coding regions were found. Nine are of one type (alpha). The other six include four different types (beta, beta', gamma and delta) which differ from each other and from the alpha genes to various degrees. Eleven of 15 genes have distinct flanking regions. Analysis of Neurospora 5S RNA showed that it consists of one principal species which matches the alpha-type gene sequence. Additional 5S species corresponding to the less abundant 5S gene types were also detected. The pattern of nucleotide substitutions between the predicted Neurospora 5S RNAs and between these and S. cerevisiae 5S RNA suggests that a particular 5S RNA secondary structure occurs in vivo and is conserved.

The lysine isoacceptor tRNAs differ in two aspects from the majority of the other mammalian tRNA species: they do not contain ribosylthymine (T) in loop IV, and a 'new' lysine tRNA, which is practically absent in non-dividing tissue, appears at elevated levels in proliferating cells. We have therefore purified the three major isoaccepting lysine tRNAs from rabbit liver and the 'new' lysine tRNA isolated from SV40-transformed mouse fibroblasts, and determined their nucleotide sequences. Our basic findings are as follows. a) The three major lysine tRNAs (species 1, 2 and 3) from rabbit liver contain 2'-O-methylribosylthymine (Tm) in place of T. tRNA1Lys and tRNA2Lys differ only by a single base pair in the middle of the anticodon stem; the anticodon sequence C-U-U is followed by N-threonyl-adenosine (t6A). TRNA3Lys has the anticodon S-U-U and contains two highly modified thionucleosides, S (shown to be 2-thio-5-carboxymethyl-uridine methyl ester) and a further modified derivative of t6 A (2-methyl-thio-N6-threonyl-adenosine) on the 3' side of the anticodon. tRNA3Lys differs in 14 and 16 positions, respectively, from the other two isoacceptors. b) Protein synthesis in vitro, using synthetic polynucleotides of defined sequence, showed that tRNA2Lys with anticodon C-U-U recognized A-A-G only, whereas tRNA3Lys, which contains thio-nucleotides in and next to the anticodon, decodes both lysine codons A-A-G and A-A-A, but with a preference for A-A-A. In a globin-mRNA-translating cell-free system from ascites cells, both lysine tRNAs donated lysine into globin. The rate and extent of lysine incorporation, however, was higher with tRNA2Lys than with tRNA3Lys, in agreement with the fact that alpha-globin and beta-globin mRNAs contain more A-A-G than A-A-A- codons for lysine. c) A comparison of the nucleotide sequences of lysine tRNA species 1, 2 and 3 from rabbit liver, with that of the 'new' tRNA4Lys from transformed and rapidly dividing cells showed that this tRNA is not the product of a new gene or group of genes, but is an undermodified tRNA derived exclusively from tRNA2Lys. Of the two dihydrouridines present in tRNA2Lys, one is found as U in tRNA4Lys; the purine next to the anticodon is as yet unidentified but is known not be t6 A. In addition we have found U, T and psi besides Tm as the first nucleoside in loop IV.

Starting from a p-aminobenzoate-requiring strain of Escherichia coli (E. coli K-12 AB3292), we have isolated mutants that can grow in the absence of p-aminobenzoate (and thus tetrahydrofolate). The following lines of evidence suggest that at least one of these mutants is capable of initiating protein synthesis without formylation of methionyl-transfer ribonucleic acid (methionyl-tRNA(fMet)). (i) tRNA isolated (and charged in vivo with [(35)S]methionine) from this mutant grown in a p-aminobenzoate-free medium contained less than 0.4% of the total methionine charged to the tRNA as formylmethionine. However, when the mutant was grown in the presence of p-aminobenzoate, 40 to 50% of the total [(35)S]methionine was detected as formylmethionine. (ii) Extracts of the mutant grown in the absence of p-aminobenzoate contained no formyl-tetrahydrofolate, but such extracts did contain formylatable methionyl-tRNA and a functional transformylase. (iii) Tetrahydrofolate-free extracts of the mutant were capable of supporting protein synthesis with viral RNA (from f2) as messenger, but the resulting synthesized proteins contained no formylmethionine, and methionine residues were detected where formylmethionine residues are normally found. In the presence of formyl-tetrahydrofolate, use of a similar extract resulted in the detection of 30 to 40% of the total polypeptide methionine as formylmethionine. (iv) Initiation of protein synthesis in vitro occurred more readily with formyl-tetrahydrofolate-free extracts of the mutant than with similar extracts prepared from the parent strain. However, in the presence of formyl-tetrahydrofolate, initiation of protein synthesis proceeded equally well with both kinds of extracts. tRNA from this mutant and another spontaneously derived mutant was found to be partially deficient in the modified nucleoside ribothymidine (rT). Analysis of extracts showed that the mutants contained decreased levels of the methylase that results in the formation of ribothymidine. In vivo studies with an independently isolated rT(-) strain suggest that the lack of rT in tRNA facilitates the growth of E. coli under conditions where protein synthesis is forced to take place without formylation.

The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs. We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo. We generated a mutant of E. coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant). This mutant tRNA has the potential to read the amber termination codon UAG. We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant). Transformation of E. coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not. Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro. Measurement of kinetic parameters for aminoacylation by E. coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics. We discuss the implications of this result on recognition of tRNAs by E. coli glutaminyl-tRNA synthetase.

Eukaryotic cytoplasmic initiator tRNAs lack the sequence G-T-Psi-C-G(A)-, which is in every tRNA of known sequence that is active in protein biosynthesis. In initiator tRNA of yeast cytoplasm, which is the only eukaryotic initiator tRNA of known sequence, this sequence is replaced by G-A-U-C-G-. We now report the sequence of a 30-nucleotide-long, 3'-terminal fragment of cytoplasmic initiator tRNA of rabbit liver obtained by specific cleavage of the tRNA at the site occupied by the modified nucleoside, 7-methyl guanosine. We show (i) that in rabbit-liver initiator tRNA also, the sequence G-T-Psi-C-G(A)- is replaced by G-A-U-C-G- and (ii) that the sequences of loop IV of both the yeast and rabbit-liver cytoplasmic initiator tRNAs are identical, A-U-C-G-m(1)-A-A-A-.

Improved solvent systems were used to crystallize two different transfer RNA species. These crystals show increased mechanical and thermal stability over crystals obtained previously from a similar system. They have sufficient stability and crystalline order to be used in x-ray crystallographic studies.

The nucleotide sequence G-T-Psi-C-G(A)- has previously been found in every tRNA of known sequence that is active in protein biosynthesis. An exception to this generalization is the recently sequenced initiator tRNA from yeast cytoplasm. It is now reported that cytoplasmic initiator tRNAs from wheat germ, rabbit liver, and sheep mammary gland also lack the G-T-Psi-C-G(A)- sequence. Thus: (i) nucleoside composition analyses show the absence of T in all these tRNAs; (ii) analyses of oligonucleotide fragments produced by T1 ribonuclease show the absence not only of the T-Psi-C-G(A)- sequence, but also of U-Psi-C-G(A)- or U-U-C-G(A)- sequences in such digests. The absence of G-T-Psi-C-G(A)- in the eukaryotic cytoplasmic initator tRNAs is, therefore, not simply due to lack of enzymatic modification of U to T.

Reproducible conditions have been developed for crystallization of transfer RNA. The conditions may be applicable to many pure transfer RNA species since identical procedures (except for initial transfer-RNA concentration) yielded good crystals from both yeast and Escherichia coli transfer RNA. These crystals, which must be kept at temperatures below about 10 degrees C and handled in vapor of controlled alcohol concentration, have been studied by x-ray crystallography. The availability of crystals of a nucleic acid opens a route for extending knowledge of the tertiary structure of transfer RNA and its relation to important biological functions.

In the early stage of bacterial translation, peptidyl-tRNAs frequently dissociate from the ribosome (pep-tRNA drop-off) and are recycled by peptidyl-tRNA hydrolase. Here, we establish a highly sensitive method for profiling of pep-tRNAs using mass spectrometry, and successfully detect a large number of nascent peptides from pep-tRNAs accumulated in Escherichia coli pthts strain. Based on molecular mass analysis, we found about 20% of the peptides bear single amino-acid substitutions of the N-terminal sequences of E. coli ORFs. Detailed analysis of individual pep-tRNAs and reporter assay revealed that most of the substitutions take place at the C-terminal drop-off site and that the miscoded pep-tRNAs rarely participate in the next round of elongation but dissociate from the ribosome. These findings suggest that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation. Peptidyl-tRNAs (pep-tRNAs) frequently dissociate from ribosome, called as pep-tRNA drop-off. But, its function remained unclear. The authors proposed a new quality control mechanism of protein synthesis by active rejection of miscoded pep-tRNAs in the early stage of translation. [ABSTRACT FROM AUTHOR]

In all species, ribosomes synthesize proteins by faithfully decoding messenger RNA (mRNA) nucleotide sequences using aminoacyl-tRNA substrates. Current knowledge of the decoding mechanism derives principally from studies on bacterial systems1. Although key features are conserved across evolution2, eukaryotes achieve higher-fidelity mRNA decoding than bacteria3. In human, changes in decoding fidelity are linked to ageing and disease and represent a potential point of therapeutic intervention in both viral and cancer treatment4–6. Here we combine single-molecule imaging and cryogenic electron microscopy methods to examine the molecular basis of human ribosome fidelity to reveal that the decoding mechanism is both kinetically and structurally distinct from that of bacteria. Although decoding is globally analogous in both species, the reaction coordinate of aminoacyl-tRNA movement is altered on the human ribosome and the process is an order of magnitude slower. These distinctions arise from eukaryote-specific structural elements in the human ribosome and in the elongation factor eukaryotic elongation factor 1A (eEF1A) that together coordinate faithful tRNA incorporation at each mRNA codon. The distinct nature and timing of conformational changes within the ribosome and eEF1A rationalize how increased decoding fidelity is achieved and potentially regulated in eukaryotic species.The reaction coordinate of aminoacyl-tRNA movement is altered on the human ribosome and the process is an order of magnitude slower compared with bacteria due to eukaryote-specific structural elements in the human ribosome and in the elongation factor eEF1A. [ABSTRACT FROM AUTHOR]

Transfer RNAs (tRNAs) can regulate cell behavior and are associated with neurological disorders. Here, we aimed to investigate the expression levels of tRNAs in oligodendrocyte precursor cells (OPCs) and their possible roles in the regulation of brain white matter injury (WMI). Newborn Sprague-Dawley rats (postnatal day 5) were used to establish a model that mimicked neonatal brain WMI. RNA-array analysis was performed to examine the expression of tRNAs in OPCs. psRNAtarget software was used to predict target mRNAs of significantly altered tRNAs. Gene ontology (GO) and KEGG were used to analyze the pathways for target mRNAs. Eighty-nine tRNAs were changed after WMI (fold change absolute ≥1.5, P  < 0.01), with 31 downregulated and 58 upregulated. Among them, three significantly changed tRNAs were identified, with two being significantly increased (chr10.trna1314-ProTGG and chr2.trna2771-ProAGG) and one significantly decreased (chr10.trna11264-GlyTCC). Further, target mRNA prediction and GO/KEGG pathway analysis indicated that the target mRNAs of these tRNAs are mainly involved in G-protein coupled receptor signaling pathways and beta-alanine metabolism, which are both related to myelin formation. In summary, the expression of tRNAs in OPCs was significantly altered after brain WMI, suggesting that tRNAs may play important roles in regulating WMI. This improves the knowledge about WMI pathophysiology and may provide novel treatment targets for WMI., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

H.G. Khorana's seminal contributions to molecular biology are well-known. He also had a lesser known but still major influence on current application of advanced vibrational spectroscopic techniques such as FTIR difference spectroscopy to explore the mechanism of bacteriorhodopsin and other integral membrane proteins. In this review, I provide a personal perspective of my collaborative research and interactions with Gobind, from 1982 to 1995 when our groups published over 25 papers together which resulted in an early picture of key features of the bacteriorhodopsin proton pump mechanism. Much of this early work served as a blueprint for subsequent advances based on combining protein bioengineering and vibrational spectroscopic techniques to study integral membrane proteins. [ABSTRACT FROM AUTHOR]

Here I will review the development of gene expression systems for production of bovine rhodopsin in the Khorana laboratory with particular focus on stable mammalian cell lines made using human embryonic kidney cells (HEK293S). The synthesis of a gene encoding bovine rhodopsin was completed in 1986. This gene was expertly designed with the built-in capacity for DNA duplex cassette replacement mutagenesis which made site-directed mutagenesis relatively straightforward. Intense effort was expended over several years in order to identify a gene expression system capable of producing rhodopsin in milligram amounts as required for biophysical studies. Mammalian expression systems, both transient and stable, were found to be the most favourable based on several criteria including receptor expression levels, correct folding and post translational processing, and capacity for purification of fully functional receptor. Transient expression using COS-1 cells was preferred for routine small-scale production of rhodopsin mutants, while HEK293S stable cell lines were used when milligram amounts of rhodopsin mutants were needed; for example, when conducting NMR studies. [ABSTRACT FROM AUTHOR]

The centenary of the birth of H. Gobind Khorana provides an auspicious opportunity to review the origins and evolution of parallel advances in biophysical methodology and molecular genetics technology used to study membrane proteins. Interdisciplinary work in the Khorana laboratory in the late 1970s and for the next three decades led to productive collaborations and fostered three subsequent scientific generations whose biophysical work on membrane proteins has led to detailed elucidation of the molecular mechanisms of energy transduction by the light-driven proton pump bacteriorhodopsin (bR) and signal transduction by the G protein–coupled receptor (GPCR) rhodopsin. This review will highlight the origins and advances of biophysical studies of membrane proteins made possible by the application of molecular genetics approaches to engineer site-specific alterations of membrane protein structures. [ABSTRACT FROM AUTHOR]

During the past decade, genetic code expansion has been proved to be a powerful tool for protein studies and engineering. As the key part, a series of orthogonal pairs have been developed to site‐specifically incorporate hundreds of noncanonical amino acids (ncAAs) into proteins by using bacteria, yeast, mammalian cells, animals, or plants as hosts. Among them, the pair of tyrosyl‐tRNA synthetase/tRNATyr from Methanococcus jannaschii and the pair of pyrrolysyl‐tRNA synthetase/tRNAPyl from Methanosarcina species are the most popular ones. Recently, other "not‐so‐popular" orthogonal pairs have started to attract attentions, because they can provide more choices of ncAA candidates and are necessary for simultaneous incorporation of multiple ncAAs into a single protein. Here, we summarize the development and applications of those "not‐so‐popular" orthogonal pairs, providing guidance for studying and engineering proteins. [ABSTRACT FROM AUTHOR]

For use in site‐specific bioorthogonal labeling of expressed G protein‐coupled receptors (GPCRs) in live cells, we developed a luciferase‐based reporter assay. The assay was used to compare amber codon suppression efficiency, receptor functionality, and efficiency of different bioorthogonal labeling chemistries. We used the assay system to compare side‐by‐side the efficiency of incorporation of three different noncanonical amino acids [4‐azido‐l‐phenylalanine (azF), cyclopropene‐l‐lysine (CpK), and trans‐cyclooct‐2‐en‐l‐lysine (TCOK)] at three different sites on a GPCR using three different genetic code expansion plasmid systems. As a model GPCR, we engineered an epitope‐tagged C‐C chemokine receptor 5 (CCR5)‐RLuc3 fusion for expression in HEK293T cells. Satisfactory incorporation of azF, CpK, and TCOK into heterologously expressed CCR5 was achieved. We also carried out cell‐based calcium mobilization assays to measure the function of the engineered CCR5, and in the same cells, we performed bioorthogonal labeling of the engineered mutants using heterobivalent compounds containing bioorthogonal tethering groups linked to either a small‐molecule fluorophore or a peptide. Favorable reaction kinetics of tetrazine‐containing compounds with CCR5 harboring TCOK was observed. However, bioorthogonal labeling in live cells of CCR5 harboring CpK with tetrazine‐containing compounds using the inverse electron demand Diels‐Alder ligation was overall slightly more efficient than other reactions tested. [ABSTRACT FROM AUTHOR]

Chloroplasts have evolved from photosynthetic cyanobacteria-like progenitors through endosymbiosis. The chloroplasts of present-day land plants have their own transcription and translation systems that show several similarities with prokaryotic organisms. A remarkable feature of the chloroplast translation system is the use of non-AUG start codons in the protein synthesis of certain genes that are evolutionarily conserved from Algae to angiosperms. However, the biological significance of such use of non-AUG codons is not fully understood. The present study was undertaken to unravel the significance of non-AUG start codons in vivo using the chloroplast genetic engineering approach. For this purpose, stable transplastomic tobacco plants expressing a reporter gene i.e. uidA (GUS) under four different start codons (AUG/UUG/GUG/CUG) were generated and β-glucuronidase (GUS) expression was compared. To investigate further the role of promoter sequences proximal to the start codon, uidA was expressed under two different chloroplast gene promoters psbA and psbC that use AUG and a non-AUG (GUG) start codons, respectively, and also showed significant differences in the DNA sequence surrounding the start codon. Further, to delineate the role of RNA editing that creates AUG start codon by editing non-AUG codons, if any, which is another important feature of the chloroplast transcription and translation system, transcripts were sequenced. In addition, a proteomic approach was used to identify the translation initiation site(s) of GUS and the N-terminal amino acid encoded when expressed under different non-AUG start codons. The results showed that chloroplasts use non-AUG start codons in combination with the translation initiation site as an additional layer of gene regulation to over-express proteins that are required at high levels due to their high rates of turnover. [ABSTRACT FROM AUTHOR]

Eukaryotic mRNAs are modified by several chemical marks which have significant impacts on mRNA biology, gene expression, and cellular metabolism as well as on the survival and development of the whole organism. The most abundant and well-studied mRNA base modifications are m6A and ADAR RNA editing. Recent studies have also identified additional mRNA marks such as m6Am, m5C, m1A and Ψ and studied their roles. Each type of modification is deposited by a specific writer, many types of modification are recognized and interpreted by several different readers and some types of modifications can be removed by eraser enzymes. Several works have addressed the functional relationships between some of the modifications. In this review we provide an overview on the current status of research on the different types of mRNA modifications and about the crosstalk between different marks and its functional consequences. [ABSTRACT FROM AUTHOR]

Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNAAla and the anticodon plays no role in charging, tRNAAla variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNAAla anticodon variants on the growth of Saccharomyces cerevisiae. Overall, 36 tRNAAla anticodon variants decreased growth in single- or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C-rich anticodons resulted in larger growth deficits than A/U-rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNAAla variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNAAla anticodon variants. [ABSTRACT FROM AUTHOR]