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T-box riboswitches are widespread bacterial regulatory noncoding RNAs that directly interact with tRNAs and switch conformations to regulate the transcription or translation of genes related to amino acid metabolism. Recent studies in Bacilli have revealed the core mechanisms of T-boxes that enable multivalent, specific recognition of both the identity and aminoacylation status of the tRNA substrates. However, in-depth knowledge on a vast number of T-boxes in other bacterial species remains scarce, although a remarkable structural diversity, particularly among pathogens, is apparent. In the present study, analysis of T-boxes that control the transcription of glycyl-tRNA synthetases from four prominent human pathogens revealed significant structural idiosyncrasies. Nonetheless, these diverse T-boxes maintain functional T-box:tRNA Gly interactions both in vitro and in vivo. Probing analysis not only validated recent structural observations, but also expanded our knowledge on the substantial diversities among T-boxes and suggest interesting distinctions from the canonical Bacilli T-boxes. Surprisingly, some glycyl T-boxes seem to redirect the T-box trajectory in the absence of recognizable K-turns or contain Stem II modules that are generally absent in glycyl T-boxes. These results consolidate the notion of a lineage-specific diversification and elaboration of the T-box mechanism and corroborate the potential of T-boxes as promising species-specific RNA targets for next-generation antibacterial compounds., (© 2024 Giarimoglou et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNA Gly and BioRNA Leu , respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNA Gly and BioRNA Leu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNA Gly /miR-7-5p exhibited greater efficiency to regulate a known target gene expression ( EGFR ) than BioRNA Leu /miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNA Gly /miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development., (© 2024 Traber et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Two types of glycyl-tRNA synthetase (GlyRS) are known, the α2 and the α2β2 GlyRSs. Both types of synthetase employ a class II catalytic domain to aminoacylate tRNAGly. In plastids and some bacteria, the α and β subunits are fused and are designated as (αβ)2 GlyRSs. While the tRNA recognition and aminoacylation mechanisms are well understood for α2 GlyRSs, little is known about the mechanisms for α2β2/(αβ)2 GlyRSs. Here we describe structures of the (αβ)2 GlyRS from Oryza sativa chloroplast by itself and in complex with cognate tRNAGly. The set of structures reveals that the U-shaped β half of the synthetase selects the tRNA in a two-step manner. In the first step, the synthetase engages the elbow and the anticodon base C35 of the tRNA. In the second step, the tRNA has rotated ∼9° toward the catalytic centre. The synthetase probes the tRNA for the presence of anticodon base C36 and discriminator base C73. This intricate mechanism enables the tRNA to access the active site of the synthetase from a direction opposite to that of most other class II synthetases., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Dnmt2, a member of the DNA methyltransferase superfamily, catalyzes the formation of 5-methylcytosine at position 38 in the anticodon loop of tRNAs. Dnmt2 regulates many cellular biological processes, especially the production of tRNA-derived fragments and intergenerational transmission of paternal metabolic disorders to offspring. Moreover, Dnmt2 is closely related to human cancers. The tRNA substrates of mammalian Dnmt2s are mainly detected using bisulfite sequencing; however, we lack supporting biochemical data concerning their substrate specificity or recognition mechanism. Here, we deciphered the tRNA substrates of human DNMT2 (hDNMT2) as tRNAAsp(GUC), tRNAGly(GCC) and tRNAVal(AAC). Intriguingly, for tRNAAsp(GUC) and tRNAGly(GCC), G34 is the discriminator element; whereas for tRNAVal(AAC), the inosine modification at position 34 (I34), which is formed by the ADAT2/3 complex, is the prerequisite for hDNMT2 recognition. We showed that the C32U33(G/I)34N35 (C/U)36A37C38 motif in the anticodon loop, U11:A24 in the D stem, and the correct size of the variable loop are required for Dnmt2 recognition of substrate tRNAs. Furthermore, mammalian Dnmt2s possess a conserved tRNA recognition mechanism., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

AaRSs (aminoacyl-tRNA synthetases) group into two ten-member classes throughout evolution, with unique active site architectures defining each class. Most are monomers or homodimers but, for no apparent reason, many bacterial GlyRSs are heterotetramers consisting of two catalytic α-subunits and two tRNA-binding β-subunits. The heterotetrameric GlyRS from Escherichia coli (EcGlyRS) was historically tested whether its α- and β-polypeptides, which are encoded by a single mRNA with a gap of three in-frame codons, are replaceable by a single chain. Here, an unprecedented X-shaped structure of EcGlyRS shows wide separation of the abutting chain termini seen in the coding sequences, suggesting strong pressure to avoid a single polypeptide format. The structure of the five-domain β-subunit is unique across all aaRSs in current databases, and structural analyses suggest these domains play different functions on α-subunit binding, ATP coordination and tRNA recognition. Moreover, the X-shaped architecture of EcGlyRS largely fits with a model for how two classes of tRNA synthetases arose, according to whether enzymes from opposite classes can simultaneously co-dock onto separate faces of the same tRNA acceptor stem. While heterotetrameric GlyRS remains the last structurally uncharacterized member of aaRSs, our study contributes to a better understanding of this ancient and essential enzyme family., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia in adults. Several studies have identified various prognostic biomarkers in CLL. In this study, we investigated the potential value of an internal fragment of the tRNAs bearing the Glycine anticodon CCC (i-tRF-GlyCCC), which is a small non-coding RNA, as a prognostic and screening biomarker in CLL. For this purpose, blood samples were collected from 90 CLL patients and 43 non-leukemic blood donors. Peripheral blood mononuclear cells (PBMCs) were isolated, total RNA was extracted and in-vitro polyadenylated, and first-strand cDNA was synthesized using an oligo-dT-adaptor primer. A real-time quantitative PCR assay was developed and applied for the quantification of i-tRF-GlyCCC in our samples. The biostatistical analysis revealed that i-tRF-GlyCCC levels are significantly lower in PBMCs of CLL patients, compared to PBMCs of non-leukemic controls, and that i-tRF-GlyCCC could be considered as a screening biomarker. Kaplan-Meier overall survival (OS) analysis revealed reduced OS for CLL patients with positive i-tRF-GlyCCC expression (P = 0.001). Multivariate Cox regression confirmed its independent unfavorable prognostic power with regard to OS. In conclusion, i-tRF-GlyCCC may constitute a promising molecular biomarker in CLL, for screening and prognostic purposes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We have previously shown that 5' halves from tRNAGlyGCC and tRNAGluCUC are the most enriched small RNAs in the extracellular space of human cell lines, and especially in the non-vesicular fraction. Extracellular RNAs are believed to require protection by either encapsulation in vesicles or ribonucleoprotein complex formation. However, deproteinization of non-vesicular tRNA halves does not affect their retention in size-exclusion chromatography. Thus, we considered alternative explanations for their extracellular stability. In-silico analysis of the sequence of these tRNA-derived fragments showed that tRNAGly 5' halves can form homodimers or heterodimers with tRNAGlu 5' halves. This capacity is virtually unique to glycine tRNAs. By analyzing synthetic oligonucleotides by size exclusion chromatography, we provide evidence that dimerization is possible in vitro. tRNA halves with single point substitutions preventing dimerization are degraded faster both in controlled nuclease digestion assays and after transfection in cells, showing that dimerization can stabilize tRNA halves against the action of cellular nucleases. Finally, we give evidence supporting dimerization of endogenous tRNAGlyGCC 5' halves inside cells. Considering recent reports have shown that 5' tRNA halves from Ala and Cys can form tetramers, our results highlight RNA intermolecular structures as a new layer of complexity in the biology of tRNA-derived fragments.

A T-box regulator or riboswitch actively monitors the levels of charged/uncharged tRNA and participates in amino acid homeostasis by regulating genes involved in their utilization or biosynthesis. It has an aptamer domain for cognate tRNA recognition and an expression platform to sense the charge state and modulate gene expression. These two conserved domains are connected by a variable linker that harbors additional secondary structural elements, such as Stem III. The structural basis for specific tRNA binding is known, but the structural basis for charge sensing and the role of other elements remains elusive. To gain new structural insights on the T-box mechanism, a molecular envelope was calculated from small angle X-ray scattering data for the Bacillus subtilis glyQS T-box riboswitch in complex with an uncharged tRNAGly. A structural model of an anti-terminated glyQS T-box in complex with its cognate tRNAGly was derived based on the molecular envelope. It shows the location and relative orientation of various secondary structural elements. The model was validated by comparing the envelopes of the wild-type complex and two variants. The structural model suggests that in addition to a possible regulatory role, Stem III could aid in preferential stabilization of the T-box anti-terminated state allowing read-through of regulated genes., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aminoacyl-tRNA synthetases are essential components of the protein translational machinery in all living species, among which the human glycyl-tRNA synthetase (hGlyRS) is of great research interest because of its unique species-specific aminoacylation properties and noncanonical roles in the Charcot-Marie-Tooth neurological disease. However, the molecular mechanisms of how the enzyme carries out its classical and alternative functions are not well understood. Here, we report a complex structure of the wild-type hGlyRS bound with tRNA(Gly) at 2.95Å. In the complex, the flexible Whep-TRS domain is visible in one of the subunits of the enzyme dimer, and the tRNA molecule is also completely resolved. At the active site, a glycyl-AMP molecule is synthesized and is waiting for the transfer of the glycyl moiety to occur. This cocrystal structure provides us with new details about the recognition mechanism in the intermediate stage during glycylation, which was not well elucidated in the previous crystal structures where the inhibitor AMPPNP was used for crystallization. More importantly, the structural and biochemical work conducted in the current and previous studies allows us to build a model of the full-length hGlyRS in complex with tRNA(Gly), which greatly helps us to understand the roles that insertions and the Whep-TRS domain play in the tRNA-binding process. Finally, through structure comparison with other class II aminoacyl-tRNA synthetases bound with their tRNA substrates, we found some commonalities of the aminoacylation mechanism between these enzymes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Recently, a growing number of reports had shown the association between mitochondrial DNA (mtDNA) sequence variants and aplastic anemia (AA). Owing to its high mutation rate, mtDNA variant had become biomarker for clinical and molecular diagnosis for AA. However, the relationship between mtDNA variant and AA was largely unknown. In this study, we reanalyzed the possible association between a "pathogenic" mutation A10055G in mt-tRNA(Gly) gene and AA, through the application of bioinformatics tool, we found that this mutation did not alter the secondary structure of tRNA(Gly), the pathogenicity scoring system indicated that the score of this mutation was only two points and belonged to a "neutral polymorphism", suggested that the role of A10055G mutation in clinical expression in AA needed to be further experimentally addressed.

D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD's invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD's chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2'-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD's activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting.

In Staphylococcus aureus, a T-box riboswitch exists upstream of the glyS gene to regulate transcription of the sole glycyl-tRNA synthetase, which aminoacylates five tRNA(Gly) isoacceptors bearing GCC or UCC anticodons. Subsequently, the glycylated tRNAs serve as substrates for decoding glycine codons during translation, and also as glycine donors for exoribosomal synthesis of pentaglycine peptides during cell wall formation. Probing of the predicted T-box structure revealed a long stem I, lacking features previously described for similar T-boxes. Moreover, the antiterminator stem includes a 42-nt long intervening sequence, which is staphylococci-specific. Finally, the terminator conformation adopts a rigid two-stem structure, where the intervening sequence forms the first stem followed by the second stem, which includes the more conserved residues. Interestingly, all five tRNA(Gly) isoacceptors interact with S. aureus glyS T-box with different binding affinities and they all induce transcription readthrough at different levels. The ability of both GCC and UCC anticodons to interact with the specifier loop indicates ambiguity during the specifier triplet reading, similar to the unconventional reading of glycine codons during protein synthesis. The S. aureus glyS T-box structure is consistent with the recent crystallographic and NMR studies, despite apparent differences, and highlights the phylogenetic variability of T-boxes when studied in a genome-dependent context. Our data suggest that the S. aureus glyS T-box exhibits differential tRNA selectivity, which possibly contributes toward the regulation and synchronization of ribosomal and exoribosomal peptide synthesis, two essential but metabolically unrelated pathways., (© 2015 Apostolidi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the "Specifier Sequence," in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNA(Gly) anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3' of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

In this report, we investigate molecular pathogenic mechanism of a diabetes-associated homoplasmic mitochondrial tRNA mutation in a Han Chinese family with maternally transmitted diabetes mellitus. Of 10 adult matrilineal relatives, 5 individuals suffered from diabetes (4 subjects with only diabetes, one subject with both diabetes and hearing impairment), while other five matrilineal relatives (one with hearing loss) had glucose intolerance. The average age at onset of diabetes in matrilineal relatives was 50 years. Molecular analysis of their mitochondrial genomes identified the novel homoplasmic T10003C mutation in the tRNA(Gly) gene belonging to haplogroup M11b. The T10003C mutation is expected to form a base-pairing (13C-22G) at the highly conserved D-stem of tRNA(Gly), thereby affecting secondary structure and function of this tRNA. A tRNA Northern analysis revealed that the T10003C mutation caused ~70% reduction in the steady-state level of tRNA(Gly). An in vivo translation analysis showed ~33% reduction in the rate of mitochondrial translation in mutant cells. Oxygen consumption analysis showed the defects in overall respiratory capacity or the ATP-linked, proton leak, and maximal respiration in mutant cells. As a result, the cellular energy deficiency contributes to the development of diabetes in subjects carrying the T10003C mutation. These data provide the first direct evidence that the tRNA(Gly) mutation might be associated with diabetes. Thus, our findings may provide new insights into the understanding of pathophysiology of maternally inherited diabetes., (Copyright © 2015 © Elsevier B.V. and Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.)

Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charges tRNA molecules with cognate amino acids for protein synthesis. Glycyl-tRNA synthetase (GlyRS) is one of the most intriguing aminoacyl-tRNA synthetases due to its divergent quaternary structure and abnormal charging properties. In the past decade, mutations of human GlyRS (hGlyRS) were also found to be associated with Charcot-Marie-Tooth disease. However, the mechanisms of traditional and alternative functions of hGlyRS are poorly understood due to a lack of studies at the molecular basis. In this study we report crystal structures of wild type and mutant hGlyRS in complex with tRNA and with small substrates and describe the molecular details of enzymatic recognition of the key tRNA identity elements in the acceptor stem and the anticodon loop. The cocrystal structures suggest that insertions 1 and 3 work together with the active site in a cooperative manner to facilitate efficient substrate binding. Both the enzyme and tRNA molecules undergo significant conformational changes during glycylation. A working model of multiple conformations for hGlyRS catalysis is proposed based on the crystallographic and biochemical studies. This study provides insights into the catalytic pathway of hGlyRS and may also contribute to our understanding of Charcot-Marie-Tooth disease., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

In Gram-positive bacteria the tRNA-dependent T box riboswitch regulates the expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes through a transcription attenuation mechanism. The Specifier domain of the T box riboswitch contains the Specifier sequence that is complementary to the tRNA anticodon and is flanked by a highly conserved purine nucleotide that could result in a fourth base pair involving the invariant U33 of tRNA. We show that the interaction between the T box Specifier domain and tRNA consists of three Watson-Crick base pairs and that U33 confers stability to the complex through intramolecular hydrogen bonding. Enhanced packing within the Specifier domain loop E motif may stabilize the complex and contribute to cognate tRNA selection., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Adaptation to the host cell environment to efficiently take-over the host cell's machinery is crucial in particular for small RNA viruses like picornaviruses that come with only small RNA genomes and replicate exclusively in the cytosol. Their Internal Ribosome Entry Site (IRES) elements are specific RNA structures that facilitate the 5' end-independent internal initiation of translation both under normal conditions and when the cap-dependent host protein synthesis is shut-down in infected cells. A longstanding issue is which host factors play a major role in this internal initiation. Here, we show that the functionally most important domain V of the poliovirus IRES uses tRNA(Gly) anticodon stem-loop mimicry to recruit glycyl-tRNA synthetase (GARS) to the apical part of domain V, adjacent to the binding site of the key initiation factor eIF4G. The binding of GARS promotes the accommodation of the initiation region of the IRES in the mRNA binding site of the ribosome, thereby greatly enhancing the activity of the IRES at the step of the 48S initiation complex formation. Moonlighting functions of GARS that may be additionally needed for other events of the virus-host cell interaction are discussed.

Although the fate of most tRNA molecules in the cell is aminoacylation and delivery to the ribosome, some tRNAs are destined to fulfill other functional roles. In addition to their central role in translation, tRNA molecules participate in processes such as regulation of gene expression, bacterial cell wall biosynthesis, viral replication, antibiotic biosynthesis, and suppression of alternative splicing. In bacteria, glycyl-tRNA molecules with anticodon sequences GCC and UCC exhibit multiple extratranslational functions, including transcriptional regulation and cell wall biosynthesis. We have determined the high-resolution structures of three glycyl-tRNA anticodon arms with anticodon sequences GCC and UCC. Two of the tRNA molecules are proteinogenic (tRNA(Gly,GCC) and tRNA(Gly,UCC)), and the third is nonproteinogenic (np-tRNA(Gly,UCC)) and participates in cell wall biosynthesis. The UV-monitored thermal melting curves show that the anticodon arm of tRNA(Gly,UCC) with a loop-closing C-A(+) base pair melts at a temperature 10 °C lower than those of tRNA(Gly,GCC) and np-tRNA(Gly,UCC). U-A and C-G pairs close the loops of the latter two molecules and enhance stem stability. Mg(2+) stabilizes the tRNA(Gly,UCC) anticodon arm and reduces the T(m) differential. The structures of the three tRNA(Gly) anticodon arms exhibit small differences among one another, but none of them form the classical U-turn motif. The anticodon loop of tRNA(Gly,GCC) becomes more dynamic and disordered in the presence of multivalent cations, whereas metal ion coordination in the anticodon loops of tRNA(Gly,UCC) and np-tRNA(Gly,UCC) establishes conformational homogeneity. The conformational similarity of the molecules is greater than their functional differences might suggest. Because aminoacylation of full-length tRNA molecules is accomplished by one tRNA synthetase, the similar structural context of the loop may facilitate efficient recognition of each of the anticodon sequences.

Uridine at the wobble position of tRNA is usually modified, and modification is required for accurate and efficient protein translation. In eukaryotes, wobble uridines are modified into 5-methoxycarbonylmethyluridine (mcm(5)U), 5-carbamoylmethyluridine (ncm(5)U) or derivatives thereof. Here, we demonstrate, both by in vitro and in vivo studies, that the Arabidopsis thaliana methyltransferase AT1G31600, denoted by us AtTRM9, is responsible for the final step in mcm(5)U formation, thus representing a functional homologue of the Saccharomyces cerevisiae Trm9 protein. We also show that the enzymatic activity of AtTRM9 depends on either one of two closely related proteins, AtTRM112a and AtTRM112b. Moreover, we demonstrate that AT1G36310, denoted AtALKBH8, is required for hydroxylation of mcm(5)U to (S)-mchm(5)U in tRNA(Gly)(UCC), and has a function similar to the mammalian dioxygenase ALKBH8. Interestingly, atalkbh8 mutant plants displayed strongly increased levels of mcm(5)U, and also of mcm(5)Um, its 2'-O-ribose methylated derivative. This suggests that accumulated mcm(5)U is prone to further ribose methylation by a non-specialized mechanism, and may challenge the notion that the existence of mcm(5)U- and mcm(5)Um-containing forms of the selenocysteine-specific tRNA(Sec) in mammals reflects an important regulatory process. The present study reveals a role in for several hitherto uncharacterized Arabidopsis proteins in the formation of modified wobble uridines.

Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA (tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm(5)U) from its precursor 5-carboxymethyluridine (cm(5)U). In this study, we report that (R)- and 5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA-Arg(UCG), and tRNA-Gly(UCC), respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm(5)U are generated from mcm(5)U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm(5)U into (S)-mchm(5)U in tRNA-Gly(UCC). These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis.

tRNAs are aminoacylated with the correct amino acid by the cognate aminoacyl-tRNA synthetase. The tRNA/synthetase systems can be divided into two classes: class I and class II. Within class I, the tRNA identity elements that enable the specificity consist of complex sequence and structure motifs, whereas in class II the identity elements are assured by few and simple determinants, which are mostly located in the tRNA acceptor stem. The tRNA(Gly)/glycyl-tRNA-synthetase (GlyRS) system is a special case regarding evolutionary aspects. There exist two different types of GlyRS, namely an archaebacterial/human type and an eubacterial type, reflecting the evolutionary divergence within this system. We previously reported the crystal structures of an Escherichia coli and of a human tRNA(Gly) acceptor stem microhelix. Here we present the crystal structure of a thermophilic tRNA(Gly) aminoacyl stem from Thermus thermophilus at 1.6Šresolution and provide insight into the RNA geometry and hydration., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We analyzed cleavage of precursor tRNAs with an LNA, 2'-OCH(3), 2'-H or 2'-F modification at the canonical (c(0)) site by bacterial RNase P. We infer that the major function of the 2'-substituent at nt -1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c(0) site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3' -endo ribose and without the H-bond donor function of the 2'-substituent. LNA and 2'-OCH(3) suppressed processing at the major aberrant m(-)(1) site; instead, the m(+1) (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c(0) and m(+1) sites had different pH profiles, with a higher Mg(2+) requirement for c(0) versus m(+1) cleavage. The strong catalytic defect for LNA and 2'-OCH(3) supports a model where the extra methylene (LNA) or methyl group (2'-OCH(3)) causes a steric interference with a nearby bound catalytic Mg(2+) during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects.

Mutations in mitochondrial 12S rRNA gene are one of the most important causes of aminoglycoside-induced and nonsyndromic hearing loss. Here we report the characterization of one Han Chinese pedigree with aminoglycoside-induced and nonsyndromic hearing loss. This Chinese family carrying the 12S rRNA A1555G mutation exhibited high penetrance and expressivity of hearing impairment. In particular, penetrances of hearing loss in this family pedigree were 43.8% and 25%, respectively, when aminoglycoside-induced hearing loss was included or excluded. Mutational analysis of entire mitochondrial genomes in this family showed the homoplasmic A1555G mutation and a set of variants belonging to haplogroup Y2. Of these, the A14693G variant occurred at the extremely conserved nucleotide (conventional position 54) of the TPsiC-loop of tRNA(Glu) and was absent in 156 Chinese controls. Nucleotides at position 54 of tRNAs are often modified, thereby contributing to the structural formation and stabilization of functional tRNAs. Thus, the structural alteration of tRNA by the A14693G variant may lead to a failure in tRNA metabolism and impair mitochondrial protein synthesis, thereby worsening mitochondrial dysfunctions altered by the A1555G mutation. Therefore, the tRNA(Glu) A14693G variant may have a potential modifier role in increasing the penetrance and expressivity of the deafness-associated A1555G mutation in this Chinese pedigree.

The tRNA(Gly)/Glycyl-tRNA synthetase system belongs to the so called 'class II' in which tRNA identity elements consist of relative few and simple motifs, as compared to 'class I' where the tRNA determinants are more complicated and spread over different parts of the tRNA, mostly including the anticodon. The determinants from 'class II' although, are located in the aminoacyl stem and sometimes include the discriminator base. There exist predominant structure differences for the Glycyl-tRNA-synthetases and for the tRNA(Gly) identity elements comparing eucaryotic/archaebacterial and eubacterial systems. We focus on comparative X-ray structure analysis of tRNA(Gly) acceptor stem microhelices from different organisms. Here, we report the X-ray structure of the human tRNA(Gly) microhelix isoacceptor G9990 at 1.18A resolution. Superposition experiments to another human tRNA(Gly) microhelix and a detailed comparison of the RNA hydration patterns show a great number of water molecules with identical positions in both RNAs. This is the first structure comparison of hydration layers from two isoacceptor tRNA microhelices with a naturally occurring base pair exchange.

The tRNA(Gly)/glycyl-tRNA synthetase (GlyRS) system belongs to the so-called 'class II aminoacyl-tRNA synthetase system' in which tRNA identity elements are assured by rather few and simple determinants mostly located in the tRNA acceptor stem. Regarding evolutionary aspects, the tRNA(Gly)/GlyRS system is a special case. There exist two different types of GlyRS, namely an archaebacterial/human type and a eubacterial type reflecting an evolutionary divergence within this system. Here we report the crystal structure of a human tRNA(Gly) acceptor stem microhelix at 1.2A resolution. The local geometric parameters of the microhelix and the water network surrounding the RNA are presented. The structure complements the previously published Escherichia coli tRNA(Gly) aminoacyl stem structure.

tRNA identity elements assure the correct aminoacylation of tRNAs by the aminoacyl-tRNA synthetases with the cognate amino acid. The tRNA(Gly)/glycyl-tRNA sythetase system is member of the so-called 'class II system' in which the tRNA determinants consist of rather simple elements. These are mostly located in the tRNA acceptor stem and in the glycine case additionally the discriminator base at position 73 is required. Within the glycine-tRNA synthetases, the archaebacterial/human and the eubacterial sytems differ with respect to their protein structures and the required tRNA identity elements, suggesting a unique evolutionary divergence. In this study, we present a comparison between the crystal structures of the eubacterial Escherichia coli and the human tRNA(Gly) acceptor stem microhelices and their surrounding hydration patterns.

tRNA identity elements determine the correct aminoacylation by the cognate aminoacyl-tRNA synthetase. In class II aminoacyl tRNA synthetase systems, tRNA specificity is assured by rather few and simple recognition elements, mostly located in the acceptor stem of the tRNA. Here we present the crystal structure of an Escherichia coli tRNA(Gly) aminoacyl stem microhelix at 2.0 A resolution. The tRNA(Gly) microhelix crystallizes in the space group P3(2)21 with the cell constants a=b=35.35 A, c=130.82 A, gamma=120 degrees . The helical parameters, solvent molecules and a potential magnesium binding site are discussed.

The major dissimilarities between the eukaryotic/archaebacterial-type and eubacterial-type glycyl-tRNA synthetase systems (GlyRS; class II aminoacyl-tRNA synthetases) represent an intriguing example of evolutionarily divergent solutions to similar biological functions. The differences in the identity elements of the respective tRNA(Gly) systems are located within the acceptor stem and include the discriminator base U73. In the present work, the human tRNA(Gly) acceptor-stem microhelix was crystallized in an attempt to analyze the structural features that govern the correct recognition of tRNA(Gly) by the eukaryotic/archaebacterial-type glycyl-tRNA synthetase. The crystals of the human tRNA(Gly) acceptor-stem helix belong to the monoclinic space group C2, with unit-cell parameters a = 37.12, b = 37.49, c = 30.38 A, alpha = gamma = 90, beta = 113.02 degrees, and contain one molecule per asymmetric unit. A high-resolution data set was acquired using synchrotron radiation and the data were processed to 1.2 A resolution.

The editing domain of alanyl-tRNA synthetase (AlaRS) contributes to high-fidelity aminoacylation by hydrolyzing (editing) the incorrect products Ser-tRNA(Ala) and Gly-tRNA(Ala) (cis-editing). The AlaX protein shares sequence homology to the editing domain of AlaRS. There are three types of AlaX proteins, with different numbers of amino-acid residues (AlaX-S, AlaX-M and AlaX-L). In this report, AlaX-M from Pyrococcus horikoshii is shown to deacylate Ser-tRNA(Ala) and Gly-tRNA(Ala) (trans-editing). The crystal structure of P. horikoshii AlaX-M has been determined at 2.7 A resolution. AlaX-M consists of an N-terminal domain (N-domain) and a C-terminal domain (C-domain). A zinc ion is coordinated by the conserved zinc-binding cluster in the C-domain, which is expected to be the enzymatic active site. The glycine-rich motif, consisting of successive conserved glycine residues in the N-domain, forms a loop (the 'glycine-rich loop'). The glycine-rich loop is located near the active site and may be involved in substrate recognition and/or catalysis.

The tRNA(Gly) and glycyl-tRNA synthetase (GlyRS) system is an evolutionary special case within the class II aminoacyl-tRNA synthetases because two divergent types of GlyRS exist: an archaebacterial/human type and an eubacterial type. The tRNA identity elements which determine the correct aminoacylation process are located in the aminoacyl domain of tRNA(Gly). To obtain further insight concerning structural investigation of the identity elements, the Escherichia coli seven-base-pair tRNA(Gly) acceptor-stem helix was crystallized. Data were collected to 2.0 A resolution using synchrotron radiation. Crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 35.35, c = 130.82 A, alpha = beta = 90, gamma = 120 degrees and two molecules in the asymmetric unit.

Plant mitochondrial genomes lack a number of tRNA genes and the corresponding tRNAs, which are nuclear-encoded, are imported from the cytosol. We show that specific import of tRNA(Gly) isoacceptors occurs in tobacco mitochondria: tRNA(Gly)(UCC) and tRNA(Gly)(CCC) are cytosolic and mitochondrial, while tRNA(Gly)(GCC) is found only in the cytosol. Exchange of sequences between tRNA(Gly)(UCC) and tRNA(Gly)(GCC) shows that the anticodon and D-domain are essential for tRNA(Gly)(UCC) import. However the reverse mutations in tRNA(Gly)(GCC) are not sufficient to promote its import into tobacco mitochondria.

The T box system regulates expression of amino acid-related genes in Gram-positive bacteria through premature termination of transcription. Synthesis of the full-length mRNA requires stabilization of an antiterminator element in the 5' untranslated leader RNA by the cognate uncharged tRNA. tRNA(Gly)-dependent antitermination of the Bacillus subtilis glyQS gene (encoding glycyl-tRNA synthetase) can be reproduced in a purified in vitro transcription system, indicating that the nascent transcript is sufficient for interaction with the tRNA. Genetic analyses previously demonstrated base pairing of a single codon in the leader RNA with the tRNA anticodon, and between the antiterminator and the tRNA acceptor end. In this study, we established conditions for specific binding of tRNA(Gly) to glyQS leader RNA generated by phage T7 RNA polymerase. Structural mapping studies revealed tRNA(Gly)-induced protection in the glyQS leader RNA at the two known sites of interaction with the tRNA, as well as at other regions between these sites. The proposed tRNA-dependent structural switch between the competing terminator and antiterminator forms of the leader RNA was demonstrated directly. Changes in tRNA(Gly) upon binding to glyQS leader RNA were detected in the anticodon loop, consistent with pairing with the specifier sequence, and in the highly conserved G19 in the D-loop, similar to effects induced by codon-anticodon interaction in the ribosome. This study provides biochemical evidence for direct interaction of tRNA(Gly) with full-length in vitro transcribed glyQS leader RNA, and an initial view of structural modulations of both RNA partners within the complex.

To elucidate the tRNA recognition sites of glycine tRNA from an extreme thermophilic and aerobic archaeon, Aeropyrum pernix K1, we examined glycylation activities using in vitro mutant glycine tRNA transcripts and recombinant A. pernix glycyl-tRNA synthetase. The recognition nucleotides were determined to be C35 and C36 of anticodon, C2-G71 and G3-C70 base-pairs of acceptor stem. However, discriminator base A73 was not recognized by glycyl-tRNA synthetase.

Methylation of tRNA at the N-1 position of guanosine to form m(1)G occurs widely in nature. It occurs at position 37 in tRNAs from all three kingdoms, and the methyltransferase that catalyzes this reaction is known from previous work of others to be critically important for cell growth in Escherichia coli and the yeast Saccharomyces cerevisiae. m(1)G is also widely found at position 9 in eukaryotic tRNAs, but the corresponding methyltransferase was unknown. We have used a biochemical genomics approach with a collection of purified yeast GST-ORF fusion proteins to show that m(1)G(9) formation of yeast tRNA(Gly) is associated with ORF YOL093w, named TRM10. Extracts lacking Trm10p have undetectable levels of m(1)G(9) methyltransferase activity but retain normal m(1)G(37) methyltransferase activity. Yeast Trm10p purified from E. coli quantitatively modifies the G(9) position of tRNA(Gly) in an S-adenosylmethionine-dependent fashion. Trm10p is responsible in vivo for most if not all m(1)G(9) modification of tRNAs, based on two results: tRNA(Gly) purified from a trm10-Delta/trm10-Delta strain is lacking detectable m(1)G; and a primer extension block occurring at m(1)G(9) is removed in trm10-Delta/trm10-Delta-derived tRNAs for all 9 m(1)G(9)-containing species that were testable by this method. There is no obvious growth defect of trm10-Delta/trm10-Delta strains. Trm10p bears no detectable resemblance to the yeast m(1)G(37) methyltransferase, Trm5p, or its orthologs. Trm10p homologs are found widely in eukaryotes and many archaea, with multiple homologs in several metazoans, including at least three in humans.

Ribosomes bypass a 50 nucleotide non-coding segment of mRNA between the two open reading frames of bacteriophage T4 gene 60 in order to synthesize a topoisomerase subunit. While nearly all ribosomes appear to initiate bypassing, only 50 % resume translation in the second open reading frame. Failure to bypass is shown here to be independent of the stop codon at the end of the first open reading frame and to be amplified by mutant variants of tRNA(Gly)(2) known to diminish bypassing efficiency. Unproductive bypassing may result from premature dissociation of peptidyl-tRNAs from ribosomes (drop-off) or resumption of translation at inappropriate sites. Assessment of the influence of factors known to induce drop-off reveals that ribosome recycling factor accounts for a small fraction of unproductive bypassing products, but none of the other known factors appear to play a significant role. Resumption of translation at inappropriate sites appears to be minimal, which suggests that spontaneous release of the peptidyl-tRNA may account for the remaining unproductive bypassing products and may be inherent to the gene 60 bypassing mechanism., (Copyright 2001 Academic Press.)

A 50-nucleotide coding gap divides bacteriophage T4 gene 60 into two open reading frames. In response to cis-acting stimulatory signals encrypted in the mRNA, the anticodon of the ribosome-bound peptidyl tRNA dissociates from a GGA codon at the end of the first open reading frame and pairs with a GGA codon 47 nucleotides downstream just before the second open reading frame. Mutations affecting ribosomal protein L9 or tRNA(Gly)(2), the tRNA that decodes GGA, alter the efficiency of bypassing. To understand the mechanism of ribosome slippage, this work analyzes the influence of these bypassing signals and mutant translational components on -1 frameshifting at G GGA and hopping over a stop codon immediately flanked by two GGA glycine codons (stop-hopping). Mutant variants of tRNA(Gly)(2) that impair bypassing mediate stop-hopping with unexpected landing specificities, suggesting that these variants are defective in ribosomal P-site codon-anticodon pairing. In a direct competition between -1 frameshifting and stop-hopping, the absence of L9 promotes stop-hopping at the expense of -1 frameshifting without substantially impairing the ability of mutant tRNA(Gly)(2) variants to re-pair with the mRNA by sub-optimal pairing. These observations suggest that L9 defects may stimulate ribosome slippage by enhancing mRNA movement through the ribosome rather than by inducing an extended pause in translation or by destabilizing P-site pairing. Two of the bypassing signals, a cis-acting nascent peptide encoded by the first open reading frame and a stemloop signal located in the 5' portion of the coding gap, stimulate peptidyl-tRNA slippage independently of the rest of the gene 60 context. Evidence is presented suggesting that the nascent peptide signal may stimulate bypassing by destabilizing P-site pairing., (Copyright 2001 Academic Press.)

We have analyzed by nucleotide analog interference mapping (NAIM) pools of precursor or mature tRNA molecules, carrying a low level of Rp-RMPalphaS (R = A, G, I) or Rp-c7-deaza-RMPalphaS (R = A, G) modifications, to identify functional groups that contribute to the specific interaction with and processing efficiency by Escherichia coli RNase P RNA. The majority of interferences were found in the acceptor stem, T arm, and D arm, including the strongest effects observed at positions G19, G53, A58, and G71. In some cases (interferences at G5, G18, and G71), the affected functional groups are candidates for direct contacts with RNase P RNA. Several modifications disrupt intramolecular tertiary contacts known to stabilize the authentic tRNA fold. Such indirect interference effects were informative as well, because they allowed us to compare the structural constraints required for ptRNA processing versus product binding. Our ptRNA processing and mature tRNA binding NAIM analyses revealed overlapping but nonidentical patterns of interference effects, suggesting that substrate binding and cleavage involves binding modes or conformational states distinct from the binding mode of mature tRNA, the product of the reaction.

We report the identification and characterization of a new mutation (ts9) in the Saccharomyces cerevisiae mitochondrial genome, which was first genetically mapped in the tRNAgly region and further identified by means of sequencing as consisting of a G to A transition at position 30 in the tRNA. The mutation causes an almost complete disappearance of mature tRNAgly, while a second mitochondrial mutation with a compensatory C to T change restores it in normal quantities; this points to the importance of the strong bond between bases 30 and 40 of the anticodon stem in the stabilization of the tRNA. In addition to resulting in a clear-cut heat-sensitive phenotype, the ts9 mutation creates a new EcoRV restriction site. Both properties were used as markers to monitor the successful (re) introduction of the mutated allele into a wild-type mitochondrial genome through biolistic transformation. The mutant frequency in the progeny as well as the correct integration of the mutated allele at its proper site demonstrate the feasibility of this method for creating and investigating specific mitochondrial tRNA mutations. The method will provide important applications for the use of yeast as a model system of human mitochondrial pathologies.

We present a systematic investigation of the thermodynamic and kinetic role of the intermolecular G292-C(75 )and G293-C(74 )Watson-Crick base-pairs in the reaction catalyzed by Escherichia coli RNase P RNA. Single turnover kinetics were analyzed for wild-type RNase P RNA and two variants with a single G to C exchange (C292 or C293), either acting on wild-type precursor tRNA (ptRNA) or derivatives carrying a complementary change at the tRNA 3'-end (G(74)CA or CG(75)A). Ground state binding of tRNA was studied using three different methods, including a novel fluorescence-based assay measuring equilibrium binding. We conclude that: (1) the role of the G293-C(74 )interaction is essentially confined to Watson-Crick base-pairing, with no indication for crucial tertiary contacts involving this base-pair; (2) the G293-C(74 )pair, although being as important for ptRNA ground state binding as G292-C(75), is much less crucial to catalytic performance than the G292-C(75) pair; (3) disruption of the G292-C(75 )base-pair results in preferential destabilization of enzyme transition-state complexes; and (4) the identity of the G292-C(75) pair, as part of the higher-order structural context consisting of coplanar G292-C(75)-A258 and G291-G259-A(76 )triples, contributes to high affinity binding of ptRNA and catalytic efficiency., (Copyright 2000 Academic Press.)

The complete nucleotide sequence of the 14,771-bp-long mitochondrial (mt) DNA of a urochordate (Chordata)-the ascidian Halocynthia roretzi-was determined. All the Halocynthia mt-genes were found to be located on a single strand, which is rich in T and G rather than in A and C. Like nematode and Mytilus edulis mtDNAs, that of Halocynthia encodes no ATP synthetase subunit 8 gene. However, it does encode an additional tRNA gene for glycine (anticodon TCT) that enables Halocynthia mitochondria to use AGA and AGG codons for glycine. The mtDNA carries an unusual tRNA(Met) gene with a TAT anticodon instead of the usual tRNA(Met)(CAT) gene. As in other metazoan mtDNAs, there is not any long noncoding region. The gene order of Halocynthia mtDNA is completely different from that of vertebrate mtDNAs except for tRNA(His)-tRNA(Ser)(GCU), suggesting that evolutionary change in the mt-gene structure is much accelerated in the urochordate line compared with that in vertebrates. The amino acid sequences of Halocynthia mt-proteins deduced from their gene sequences are quite different from those in other metazoans, indicating that the substitution rate in Halocynthia mt-protein genes is also accelerated.

Using tRNAscan-SE and FAStRNA we have identified four tRNA genes in the delayed early region of the bacteriophage D3 genome (GenBank accession No. AF077308). These are specific for methionine (AUG), glycine (GGA), asparagine (AAC), and threonine (ACA). The D3 Thr- and Gly-tRNAs recognize codons, which are rarely used in Pseudomonas aeruginosa and presumably, influence the rate of translation of phage proteins. BLASTN searches revealed that the D3 tRNA genes have homology to tRNA genes from Gram-positive bacteria. Analysis of codon usage in the 91 ORFs discovered in D3 indicates patterns of codon usage reminiscent of Escherichia coli or P. aeruginosa.

Amino acid assignments of metazoan mitochondrial codons AGA/AGG are known to vary among animal species; arginine in Cnidaria, serine in invertebrates and stop in vertebrates. We recently found that in the mitochondria of the ascidian Halocynthia roretzi these codons are exceptionally used for glycine, and postulated that they are probably decoded by a tRNA(UCU). In order to verify this notion unambig-uously, we determined the complete RNA sequence of the mitochondrial tRNA(UCU) presumed to decode codons AGA/AGG in the ascidian mitochondria, and found it to have an unidentified U derivative at the anticodon first position. We then identified the amino acids attached to the tRNA(U*CU), as well as to the conventional tRNAGly(UCC) with an unmodified U34, in vivo. The results clearly demonstrated that glycine was attached to both tRNAs. Since no other tRNA capable of decoding codons AGA/AGG has been found in the mitochondrial genome, it is most probable that this tRNA(U*CU) does actually translate codons AGA/AGG as glycine in vivo. Sequencing of tRNASer(GCU), which is thought to recognize only codons AGU/AGC, revealed that it has an unmodified guanosine at position 34, as is the case with vertebrate mitochondrial tRNASer(GCU) for codons AGA/AGG. It was thus concluded that in the ascidian, codons AGU/AGC are read as serine by tRNASer(GCU), whereas AGA/AGG are read as glycine by an extra tRNAGly(U*CU). The possible origin of this unorthodox genetic code is discussed.

In potato ( Solanum tuberosum ) mitochondria, about two-thirds of the tRNAs are encoded by the mitochondrial genome and one-third is imported from the cytosol. In the case of tRNAGly isoacceptors, a mitochondrial-encoded tRNAGly(GCC) was found in potato mitochondria, but this is likely to be insufficient to decode the four GGN glycine codons. In this work, we identified a cytosolic tRNAGly(UCC), which was found to be present in S.tuberosum mitochondria. The cytosolic tRNAGly(CCC) was also present in mitochondria, but to a lesser extent. By contrast, the cytosolic tRNAGly(GCC) could not be detected in mitochondria. This selective import of tRNAGly isoacceptors into S. tuberosum mitochondria raises further questions about the mechanism under-lying the specificity of the import process.

The tRNA glycylation system is amongst the most complex aminoacylation systems since neither the oligomeric structure of the enzymes nor the discriminator base in tRNAs are conserved in the phylae. To understand better this structural diversity and its functional consequences, the prokaryotic glycylation system from Thermus thermophilus, an extreme thermophile, was investigated and its structural and functional inter-relations with those of other origins analyzed. Alignments of the protein sequence of the dimeric thermophilic glycyl-tRNA synthetase (Gly-tRNA synthetase) derived from its gene with sequences of other dimeric Gly-tRNA synthetases revealed an atypical character of motif 1 in all these class 2 synthetases. Interestingly, the sequence of the prokaryotic thermophilic enzyme resembles eukaryotic and archaebacterial Gly-tRNA synthetases, which are all dimeric, and diverges drastically from the tetrameric enzymes from other prokaryotes. Cross aminoacylations with tRNAs and synthetases of different origins provided information about functional interrelations between the glycylation systems. Efficient glycylations involving partners from T. thermophilus and Escherichia coli showed conservation of the recognition process in prokaryotes despite strong structural variations of the synthetases. However, Gly-tRNA synthetase from T. thermophilus acylates eukaryotic tRNA(Gly) while the charging ability of the E. coli enzyme is restricted to prokaryotic tRNA(Gly). A similar behaviour is found in eukaryotic systems where the restricted species specificity for tRNA glycylation of mammalian Gly-tRNA synthetase contrasts with the relaxed specificity of the yeast enzyme. The consensus sequence of the tRNAs charged by the various Gly-tRNA synthetases reveals conservation of only G1-C72 in the acceptor arm, C35 and C36 in the anticodon, and the (G10-Y25)-G45 triplet involved in tRNA folding. Conservation of these nucleotides indicates their key role in glycylation and suggests that they were part of the ancestral glycine identity set. These features are discussed in the context of the phylogenic connections between prokaryotes, eukaryotes, and archaebacteria, and of the particular place of T. thermophilus in this phylogeny.

Escherichia coli tRNACys contains an unusual G15.G48 tertiary base pair that is important for recognition and aminoacylation by cysteine tRNA synthetase. This G15.G48 tertiary base pair has a distinctive chemical modification signature that suggests an N2.N3 base pairing. The N2.N3 pairing of a G.G base pair has not been described in any existing RNA structures. Identification of the structural determinant of G15.G48 is of fundamental importance for understanding the formation of an RNA tertiary base pair, as well as the role of RNA tertiary structure in tRNA recognition. We show here that the structural determinant for G15.G48 is an A13.A22 mismatch in the dihydrouridine stem. Introduction of A13.A22 to an unrelated tRNA confers the distinctive chemical modification signature of G15. G48 while substitution of A13.A22 eliminates this signature. The relationship between G15.G48 and A13.A22 enables the unrelated tRNA to be efficiently recognized by cysteine tRNA synthetase. Modeling studies show that A13.A22 has the potential to form a base triple with A46, which is directly connected to G48 in the G15.G48 base pair. The proposed A13.A22.A46 base triple provides a framework for understanding how two RNA structural elements may be related to each other in playing an important role in tRNA aminoacylation.

Glycyl-tRNA synthetase (GlyRS) is an unusual aminoacyl-tRNA synthetase because it varies in its quarternary structure between organisms; Escherichia coli GlyRS is an alpha2beta2 tetramer, whereas those of Thermus thermophilus and yeast are alpha2 dimers. In contrast, the tRNA(Gly) sequence is virtually identical in E. coli and T. thermophilus but very different in yeast. In this study, we examined the molecular recognition of tRNA(Gly) by three widely diverged GlyRSs using in vitro tRNA transcripts. Mutation studies showed that the discriminator base at position 73, the second base-pair, C2 x G71, in the acceptor stem, and the anticodon nucleotides, C35 and C36, contribute to the specific aminoacylation of all three GlyRSs, the discriminator base differing between prokaryotes (U73) and eukaryotes (A73). However, we found differences between yeast and two bacteria around the second base-pair in the acceptor stem. The first base-pair, G1 x C72, is important for glycylation in E. coli and T. thermophilus, whereas the third base-pair, G3 x C70, is important for glycylation in yeast. These findings indicate that despite such large differences of the two prokaryotic GlyRSs, tRNA(Gly) identity has been essentially conserved in prokaryotes, and that there are also differences in the acceptor stem recognition between prokaryotes and yeast. The clear separation between prokaryotes and yeast is retained in the identity element location, whereas the apparent diversity of the two prokaryotic enzymes does not reflect on the tRNA recognition.

Glycyl-tRNA synthetase (GlyRS) is an unusual aminoacyl-tRNA synthetase because it varies in its quarternary structure between organisms; Escherichia coli GlyRS is an alpha 2 beta 2 tetramer, whereas those of Thermus thermophilus and yeast are alpha 2 dimers. In contrast, the tRNA(Gly) sequence is virtually identical in E. coli and T. thermophilus but very different in yeast. In this study, we examined the molecular recognition of tRNA(Gly) by three widely diverged GlyRSs using in vitro tRNA transcripts. The results obtained in the mutation studies indicate that despite such large differences of the two prokaryotic GlyRSs, tRNA(Gly) identity has been essentially conserved in prokaryotes, and that there are also differences in the acceptor stem recognition between prokaryotes and yeast. The clear separation between prokaryotes and yeast is retained in the identity element location, whereas the apparent diversity of the two prokaryotic enzymes does not reflect on the tRNA recognition.

RNA minihelices which reconstruct the 12 base pair acceptor-T psi C domains of transfer RNAs interact with their cognate tRNA synthetases. These substrates lack the anticodons of the genetic code and, therefore, cannot participate in steps of protein synthesis subsequent to aminoacylation. We report here that expression in Escherichia coli of either of two minihelices, each specific for a different amino acid, inhibited cell growth. Inhibition appears to be due to direct competition between the minihelix and its related tRNA for binding to their common synthetase. This competition, in turn, sharply lowers the pool of the specific charged tRNA for protein synthesis. Inhibition is relieved by single nucleotide changes which disrupt the minihelix-synthetase interaction. The results suggest that sequence-specific RNA minihelix substrates bind to cognate synthetases in vivo and can, in principle, act as cell growth regulators. Naturally occurring non-tRNA substrates for aminoacylation may serve a similar purpose.

The recognition of transfer RNAs (tRNAs) by aminoacyl tRNA synthetases is a critical step in establishing the fidelity of translation. For E. coli cysteine tRNA synthetase, recognition of tRNA(Cys) and discrimination from all other tRNAs is based on the U73 discriminator base, the GCA anticodon, and a G15:G48 tertiary base pair. While the discriminator base and the anticodon sequence are often used by many synthetases as the determinants for tRNA recognition, the dependence on a tertiary interaction in the cognate tRNA for recognition is unique to the cysteine enzyme. Here the structural basis for recognition at the G15:G48 tertiary interaction of E. coli tRNA(Cys) is explored by structural modeling, chemical modifications, and kinetic analysis. The results established an unusual RNA tertiary interaction that provides a plausible mechanism for recognition by cysteine tRNA synthetase.

The nucleotide sequence of 1029 bp BamHI-fragment of leek chloroplast DNA (Allium porrum, fam. Liliaceae) has been determined. The fragment contains the 3'-terminal part of the tRNA-Gly (UCC) gene, the tRNA-Arg (UCU) gene and the 3'-terminal domain of the CF1ATPase alpha-subunit gene. The gene arrangement and the nucleotide sequence of this fragment are similar to those of the tobacco chloroplast DNA but differ significantly from that of other monocots in which the region containing these genes underwent extensive recombination.