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The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aside from providing adaptive immunity, type I CRISPR-Cas was recently unearthed to employ a noncanonical RNA guide (CreA) to transcriptionally repress an RNA toxin (CreT). Here, we report that, for most archaeal and bacterial CreTA modules, the creA gene actually carries two flanking 'CRISPR repeats', which are, however, highly divergent and degenerated. By deep sequencing, we show that the two repeats give rise to an 8-nt 5' handle and a 22-nt 3' handle, respectively, i.e., the conserved elements of a canonical CRISPR RNA, indicating they both retained critical nucleotides for Cas6 processing during divergent degeneration. We also uncovered a minimal CreT toxin that sequesters the rare transfer RNA for isoleucine, tRNAIleCAU, with a six-codon open reading frame containing two consecutive AUA codons. To fully relieve its toxicity, both tRNAIleCAU overexpression and supply of extra agmatine (modifies the wobble base of tRNAIleCAU to decipher AUA codons) are required. By replacing AUA to AGA/AGG codons, we reprogrammed this toxin to sequester rare arginine tRNAs. These data provide essential information on CreTA origin and for future CreTA prediction, and enrich the knowledge of tRNA-sequestering small RNAs that are employed by CRISPR-Cas to get addictive to the host., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Here we report that RNase P is required for the initial separation of all seven valine tRNAs from three distinct polycistronic transcripts (valV valW, valU valX valY lysY and lysT valT lysW valZ lysY lysZ lysQ). Particularly significant is the mechanism by which RNase P processes the valU and lysT polycistronic transcripts. Specifically, the enzyme initiates processing by first removing the Rho-independent transcription terminators from the primary valU and lysT transcripts. Subsequently, it proceeds in the 3' → 5' direction generating one pre-tRNA at a time. Based on the absolute requirement for RNase P processing of all three primary transcripts, inactivation of the enzyme leads to a > 4-fold decrease in the levels of both type I and type II valine tRNAs. The ability of RNase P to initiate tRNA processing at the 3' ends of long primary transcripts by endonucleolytically removing the Rho-independent transcription terminator represents a previously unidentified function for the enzyme, which is responsible for generating the mature 5' termini of all 86 E. coli tRNAs. RNase E only plays a very minor role in the processing of all three valine polycistronic transcripts., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNA(Arg)ICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNA(Arg)CCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNA(Arg)CCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNA(Arg) and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNA(Arg)ACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNA(Arg)ACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNA(Arg)CCG and tadA, and then acquired a new tRNA(Arg)UCG. This permitted further tRNA(Arg)ACG mutations with tRNA(Arg)GCG or its disappearance, leaving a single tRNA(Arg)UCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.

RNA editing changes the coding/decoding information relayed by transcripts via nucleotide insertion, deletion, or conversion. Editing of tRNA anticodons by deamination of adenine to inosine is used both by eukaryotes and prokaryotes to expand the decoding capacity of individual tRNAs. This limits the number of tRNA species required for codon-anticodon recognition. We have identified the Arabidopsis thaliana gene that codes for tRNA adenosine deaminase arginine (TADA), a chloroplast tRNA editing protein specifically required for deamination of chloroplast (cp)-tRNAArg(ACG) to cp-tRNAArg(ICG). Land plant TADAs have a C-terminal domain similar in sequence and predicted structure to prokaryotic tRNA deaminases and also have very long N-terminal extensions of unknown origin and function. Biochemical and mutant complementation studies showed that the C-terminal domain is sufficient for cognate tRNA deamination both in vitro and in planta. Disruption of TADA has profound effects on chloroplast translation efficiency, leading to reduced yields of chloroplast-encoded proteins and impaired photosynthetic function. By contrast, chloroplast transcripts accumulate to levels significantly above those of wild-type plants. Nevertheless, absence of cp-tRNAArg(ICG) is compatible with plant survival, implying that two out of three CGN codon recognition occurs in chloroplasts, though this mechanism is less efficient than wobble pairing.

A synthetic genetic array was used to identify lethal and slow-growth phenotypes produced when a mutation in TRM6, which encodes a tRNA modification enzyme subunit, was combined with the deletion of any non-essential gene in Saccharomyces cerevisiae. We found that deletion of the REX1 gene resulted in a slow-growth phenotype in the trm6-504 strain. Previously, REX1 was shown to be involved in processing the 3' ends of 5S rRNA and the dimeric tRNA(Arg)-tRNA(Asp). In this study, we have discovered a requirement for Rex1p in processing the 3' end of tRNA(i)(Met) precursors and show that precursor tRNA(i)(Met) accumulates in a trm6-504 rex1Delta strain. Loss of Rex1p results in polyadenylation of its substrates, including tRNA(i)(Met), suggesting that defects in 3' end processing can activate the nuclear surveillance pathway. Finally, purified Rex1p displays Mg(2+)-dependent ribonuclease activity in vitro, and the enzyme is inactivated by mutation of two highly conserved amino acids.

High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNAAsp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA4Arg. This tRNA has a sequence strikingly similar to that of tRNAAsp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA4Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA4Arg into an aspartate acceptor, as efficient as tRNAAsp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA4Arg. The latent aspartate acceptor capacity in a contemporary tRNAArg leads to the proposal of an evolutionary link between tRNA4Arg and tRNAAsp genes.

Here we report the construction of a yeast genetic screen designed to identify essential residues in tRNA(Arg). The system consists of a tRNA(Arg) knockout strain and a set of vectors designed to rescue and select for variants of tRNA(Arg). By plasmid shuffling we selected inactive tRNA mutants that were further analyzed by northern blotting. The mutational analysis focused on the tRNA D and anticodon loops that contact the aminoacyl-tRNA synthetase. The anticodon triplet was excluded from the analysis because of its role in decoding the Arg codons. Most of the inactivating mutations are residues involved in tertiary interactions. These mutations had dramatic effects on tRNA(Arg) abundance. Other inactivating mutations were located in the anticodon loop, where they did not affect transcription and aminoacylation but probably altered interaction with the translation machinery. No lethal effects were observed when residues 16, 20 and 38 were individually mutated, despite the fact that they are involved in sequence-specific interactions with the aminoacyl-tRNA synthetase. However, the steady-state levels of the aminoacylated forms of U20A and U20G were decreased by a factor of 3.5-fold in vivo. This suggests that, unlike in the Escherichia coli tRNA(Arg):ArgRS system where residue 20 (A) is a major identity element, in yeast this position is of limited consequence.

tRNA 3' processing endoribonuclease (3' tRNase) is an enzyme responsible for the removal of a 3' trailer from precursor tRNA (pre-tRNA). We purified approximately 85 kDa 3' tRNase from pig liver and determined its partial sequences. BLAST search of them suggested that the enzyme was the product of a candidate human prostate cancer susceptibility gene, ELAC2, the biological function of which was totally unknown. We cloned a human ELAC2 cDNA and expressed the ELAC2 protein in Escherichia coli. The recombinant ELAC2 was able to cleave human pre-tRNA(Arg) efficiently. The 3' tRNase activity of the yeast ortholog YKR079C was also observed. The C-terminal half of human ELAC2 was able to remove a 3' trailer from pre-tRNA(Arg), while the N-terminal half failed to do so. In the human genome exists a gene, ELAC1, which seems to correspond to the C-terminal half of 3' tRNase from ELAC2. We showed that human ELAC1 also has 3'-tRNase activity. Furthermore, we examined eight ELAC2 variants that seem to be associated with the occurrence of prostate cancer for 3'-tRNase activity. Seven ELAC2 variants which contain one to three amino acid substitutions showed efficient 3'-tRNase activities, while one truncated variant, which lacked a C-terminal half region, had no activity.

The expression of the proUK gene was improved by the coexpression of the argU gene cloned in a moderate copy number vector. As the proUK gene contains 2% AGG/AGA codons, which is much higher than the normal frequency in E. coli, about 0.14%-0.21%, the argU gene cloned in a multicopy plasmid was coexpressed with the proUK expression vector in our experiments. In E. coli strain BL21(DE3), IPTG is known to induce the expression of T7 RNA polymerase gene and this enzyme can transcribe the proUK gene under the control of the T7 promoter leading to expression of proUK. To replace IPTG by a cheaper alternative on a large scale, we constructed a plasmid in which the vgb promoter--which is known to be activated by the onset of hypoxic conditions--controls the T7RNA polymerase gene expression. Low oxygen conditions were then used to activate the vgb promoter causing T7RNA polymerase gene expression and finally leading to the expression of proUK as inactive inclusion bodies. Our experiments on a large scale in a bioreactor show that the expression of proUK accounts for about 30% of total protein after about 6 h of anaerobic cultivation, so the presented model represents an economical alternative to IPTG induction.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can recognize and cleave any target RNA that forms a precursor tRNA-like complex with another RNA. Various sets of RNA molecules were tested to identify the smallest RNA that can direct target RNA cleavage by 3' tRNase. A 3' half tRNAArgwas cleaved efficiently by 3' tRNase in the presence of small 5' half tRNAArgvariants, the D stem-loop region of which was partially deleted. Remarkably, 3' tRNase also cleaved the 3' half tRNAArgin the presence of a 7 nt 5' tRNAArg composed only of the acceptor stem region with a catalytic efficiency comparable with that of cleavage directed by an intact 5' half tRNAArg. The catalytic efficiency of cleavage directed by the heptamer decreased as the stability of the T stem-loop structures of 3' half tRNAArg variants decreased. No heptamer-directed cleavage of a 3' half tRNAArg without T stem base pairs was detected. A heptamer also directed cleavage of an HIV-1 RNA containing a stable hairpin structure. These findings suggest that in the presence of an RNA heptamer, 3' tRNase can discriminate and eliminate target RNAs that possess a stable hairpin adjacent to the heptamer binding sequence from a large complex RNA pool.

Gene cloning, overproduction and an efficient purification protocol of yeast arginyl-tRNA synthetase (ArgRS) as well as the interaction patterns of this protein with cognate tRNAArgand non-cognate tRNAAspare described. This work was motivated by the fact that the in vitro transcript of tRNAAspis of dual aminoacylation specificity and is not only aspartylated but also efficiently arginylated. The crystal structure of the complex between class II aspartyl-tRNA synthetase (AspRS) and tRNAAsp, as well as early biochemical data, have shown that tRNAAspis recognized by its variable region side. Here we show by footprinting with enzymatic and chemical probes that transcribed tRNAAspis contacted by class I ArgRS along the opposite D arm side, as is homologous tRNAArg, but with idiosyncratic interaction patterns. Besides protection, footprints also show enhanced accessibility of the tRNAs to the structural probes, indicative of conformational changes in the complexed tRNAs. These different patterns are interpreted in relation to the alternative arginine identity sets found in the anticodon loops of tRNAArgand tRNAAsp. The mirror image alternative interaction patterns of unmodified tRNAAspwith either class I ArgRS or class II AspRS, accounting for the dual identity of this tRNA, are discussed in relation to the class defining features of the synthetases. This study indicates that complex formation between unmodified tRNAAspand either ArgRS and AspRS is solely governed by the proteins.

Mammalian tRNA 3'processing endoribonuclease (3'tRNase) removes 3'extra nucleotides after the discriminator from tRNA precursors. Here I examined how the length of a 3'trailer and the nucleotides on each side of the cleavage site affected 3'processing efficiency. I performed in vitro 3'processing reactions of pre-tRNAArgs with various 3'trailers or various discriminator nucleotides using 3'tRNase purified from mouse FM3A cells or pig liver. On the whole, the efficiency of pre- tRNAArg3'processing by mammalian 3'tRNase decreased as the 3'trailer became longer, except in the case of a 3'trailer composed of CC, CCA or CCA plus 1 or 2 nucleotides, which was not able to be removed at all. The distribution of 3'trailer lengths deduced from mammalian nuclear tRNA genomic sequences reflects this property of 3'tRNase. The cleavage efficiency of pre-tRNAArgs varied depending on the 5'end nucleotide of a 3'trailer in the order G approximately A > U > C. This effect of the 5'end nucleotide was independent of the discriminator nucleotides. The distribution of the 5'end nucleotides of mammalian pre-tRNA 3'trailers reflects this differential 3'processing efficiency.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can recognize pre-tRNA-like complexes between a 5' half tRNA(Arg) and a 3' half tRNA(Arg) with a 3' trailer, and can cleave the 3' half tRNA(Arg) after the discriminator nucleotide (Nashimoto M., 1996, RNA, 2:523-534). 3' tRNase cleaved the 3' half tRNA(Arg) in the presence of a 7-nt 5' tRNA(Arg) composed only of the acceptor stem region with a catalytic efficiency comparable to that of cleavage directed by the intact 5' half tRNA(Arg). The catalytic efficiency of cleavage directed by the heptamer decreased as the stability of the T stem-loop structures of 3' half tRNA(Arg) variants decreased. No heptamer-directed cleavage of a 3' half tRNA(Arg) was detected in the absence of T-stem base pairs. This strategy for targeted RNA cleavage using an RNA heptamer was applied to the cleavage of two target RNAs from HIV-1. An HIV-1 RNA containing a stable hairpin structure corresponding to the T stem-loop was cleaved efficiently, whereas no cleavage of a second HIV-1 target without any stable hairpin structure was detected. In this method, an RNA heptamer can direct efficient RNA cleavage with a higher specificity than expected.

Arginyl-tRNA synthetase (ArgRS) catalyses AMP- and PPi-independent deacylation of Arg-tRNAArg in the presence of cysteine. A dipeptide, Arg-Cys, is a product of this deacylation reaction. Similar reaction with homocysteine yields Arg-Hcy. Arginine is a noncompetitive inhibitor of the cysteine-dependent deacylation which indicates that cysteine binds to the enzyme-Arg-tRNAArg complex at a site separate from the arginine binding site. In the presence of arginine, [14C]Arg-tRNAArg is deacylated at a rate similar to the rate of its spontaneous deacylation in solution and [14C]arginine is a product. Experiments with cysteine derivatives indicate that the -SH group is essential for the reaction whereas -NH2 and -COOH groups are not. Thioesters of arginine are formed with 3-mercaptopropionic acid, N-acetyl-L-cysteine and dithiothreitol. These data suggest that formation of the dipeptide Arg-Cys involves a thioester intermediate, S-(L-arginyl)-L-cysteine, which is not observed because of the rapid rearrangement to form a stable peptide bond. Facile intramolecular reaction results from the favorable geometric arrangement of the alpha-amino group of cysteine with respect to the thioester formed in the initial reaction. Similar reactions, yielding Ile-Cys and Val-Cys, are catalyzed by isoleucyl- and valyl-tRNA synthetases, respectively.

The spermidine-dependent, sequence-specific endoribonuclease (RNase 65) activities in mammalian cell extracts require both protein and 3' truncated tRNA, species of which direct their substrate sequence specificity. Computer analysis for searching possible base pairing between substrate RNAs and their corresponding 3' truncated tRNA, suggested a unified model for substrate recognition mechanism, in which a four-nucleotide (nt) sequence in the target tRNAs 1 nt upstream of their cleavage site, base pairs with the 5' terminal 4 nt sequence of their corresponding 3' truncated tRNA. This model was supported by experiments with several RNA substrates containing a substituted nucleotide in the target 4 nt sequence. In this model, the tRNA substrates and their corresponding 3' truncated tRNA form a complex resembling a 5' processed tRNA precursor containing a 3' trailer, suggesting that the protein component of RNase 65 is identical to tRNA 3' processing endoribonuclease (3' tRNase). Actually, 3' tRNase purified from pig liver cleaved the target RNAs at the expected sites only in the presence of their corresponding 3' truncated tRNA. These results show that the 3' tRNase can be converted to 4 nt specific RNA cutters using the 3' truncated tRNAs.

In vitro synthesized 5' portions of mouse 18S rRNA are cleaved efficiently at a specific site in partially purified extracts of mouse FM3A cells and several mouse tissues. This activity is composed of both protein and RNA, and can be reconstituted with the protein component in micrococcal nuclease-treated extracts and the RNA component in phenol-treated ones. The RNA component of about 65 nucleotides with the complementing activity was purified from total RNA in the partially purified FM3A cell extracts by polyacrylamide gel electrophoreses. Chemical sequencing of this RNA elucidated that it is tRNAArg lacking nine nucleotides from its 3' terminus. Ribonuclease H treatment directed by deoxyoligonucleotides complementary to tRNAArg completely abolishes the cleavage activity, supporting the above conclusion.