On the role of an unusual tRNAGly isoacceptor in Staphylococcus aureus

In the available Staphylococcus aureus genomes, four different genes have been annotated to encode tRNA^Gly isoacceptors. Besides their prominent role in protein synthesis, some of them also participate in the formation of pentaglycine bridges during cell wall synthesis. However, until today, it is not known how many and which of them are actually involved in this essential procedure. In the present study we identified, apart from the four annotated tRNA^Gly genes, a putative pseudogene which encodes and expresses an unusual fifth tRNA^Gly isoacceptor in S. aureus (as detected via RT-PCR and subsequent direct sequencing analysis). All the in vitro transcribed tRNA^Gly molecules (including the “pseudogene-encoded” tRNA^Gly) can be efficiently aminoacylated by the recombinant S. aureus glycyl-tRNA synthetase. Furthermore, bioinformatic analysis suggests that the “pseudo”-tRNA^Gly(UCC) identified in the present study and two of the annotated isoacceptors bearing the same anticodon carry specific sequence elements that do not favour the strong interaction with EF-Tu that proteinogenic tRNAs would promote. This observation was verified by the differential capacity of Gly-tRNA^Gly molecules to form ternary complexes with activated S. aureus EF-Tu·GTP. These tRNA^Gly molecules display high sequence similarities with their S. epidermidis orthologs which also actively participate in cell wall synthesis. Both bioinformatic and biochemical data suggest that in S. aureus these three glycylated tRNA^Gly isoacceptors that are weak EF-Tu binders, possibly escape protein synthesis and serve as glycine donors for the formation of pentaglycine bridges that are essential for stabilization of the staphylococcal cell wall.     

The characterization of the RNAs and aminoacyl-tRNA synthetases of the blue-green alga, Anacystis nidulans

The tRNA and aminoacyl-tRNA synthetases of the blue-green alga, Anacystis nidulans have been isolated and studied. The distribution of some algal tRNA species on BD-cellulose chromatography has been determined. One tRNA^Met species has been isolated in 80% purity by a single chromatography on a BD-cellulose column developed with a modified salt gradient. The number of different tRNA isoacceptors for Met, Ser, and Leu has been ascertained by RPC-5 chromatography. The recognition of algal tRNAs by the homologous algal synthetase preparation as well as the heterologous Escherichia coli preparation was studied by the aminoacylation tests. Since all of the isoaccepting species of the tRNAs tested behaved almost identically in presence of the two enzyme preparations, a conservation of the recognition site during the evolutionary divergence of bacteria and algae is strongly suggested.     

Comparative X-ray structure analysis of human and Escherichia coli tRNA(Gly) acceptor stem microhelices

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.     

Recognition of tRNAGln by Helicobacter pylori GluRS2—a tRNAGln-specific glutamyl-tRNA synthetase

Accurate aminoacylation of tRNAs by the aminoacyl-tRNA synthetases (aaRSs) plays a critical role in protein translation. However, some of the aaRSs are missing in many microorganisms. Helicobacter pylori does not have a glutaminyl-tRNA synthetase (GlnRS) but has two divergent glutamyl-tRNA synthetases: GluRS1 and GluRS2. Like a canonical GluRS, GluRS1 aminoacylates tRNA^Glu1 and tRNA^Glu2. In contrast, GluRS2 only misacylates tRNA^Gln to form Glu-tRNA^Gln. It is not clear how GluRS2 achieves specific recognition of tRNA^Gln while rejecting the two H. pylori tRNA^Glu isoacceptors. Here, we show that GluRS2 recognizes major identity elements clustered in the tRNA^Gln acceptor stem. Mutations in the tRNA anticodon or at the discriminator base had little to no impact on enzyme specificity and activity.     

Crystal structure of a human tRNA(Gly) microhelix at 1.2A resolution

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 1.2 Å 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.     

The crystal structure of a Thermus thermophilus tRNA acceptor stem microhelix at 1.6 Å resolution

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 1.6 Å resolution and provide insight into the RNA geometry and hydration.     

A yeast arginine specific tRNA is a remnant aspartate acceptor

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 tRNA^Asp 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, tRNA₄^Arg. This tRNA has a sequence strikingly similar to that of tRNA^Asp 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, tRNA₄^Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA₄^Arg into an aspartate acceptor, as efficient as tRNA^Asp, 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 tRNA₄^Arg. The latent aspartate acceptor capacity in a contemporary tRNA^Arg leads to the proposal of an evolutionary link between tRNA₄^Arg and tRNA^Asp genes.     

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon–anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the ‘GTP’- and ‘GDP’-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.     

Processing of plant mitochondrial tRNAGly and tRNASer(GCU) is independent of RNA editing

The genes encoding pea and potato mitochondrial tRNA^Gly and pea mitochondrial tRNA^Ser(GCU) were analyzed with particular respect to their expression. Secondary-structure models deduced from the identical potato and pea tRNA^Gly gene sequences revealed A₇:C₆₆ mismatches in the seventh base pair at the base of the acceptor stems of both tRNAs. Sequence analyses of tRNA^Gly cDNA clones showed that these mispairings are not corrected by C₆₆ to U₆₆ conversions, as observed in plant mitochondrial tRNA^Phe. Likewise, a U₆:C₆₇ mismatch identified in the acceptor stem of the pea tRNA^Ser(GCU) is not altered by RNA editing to a mismatched U:U pair, which is created by RNA editing in Oenothera mitochondrial tRNA^Cys. In vitro processing reactions with the respective tRNA^Gly and tRNA^Ser(GCU) precursors show that such conversions are not necessary for 5′ and 3′ end maturation of these tRNAs. These results demonstrate that not all C:A (A:C) or U:C (C:U) mismatches in double-stranded regions of tRNAs are altered by RNA editing. An RNA editing event in plant mitochondrial tRNAs is thus not generally indicated by the presence of a mismatch but may depend on additional parameters.     

tRNAGlu Increases the Affinity of Glutamyl-tRNA Synthetase for Its Inhibitor Glutamyl-Sulfamoyl-Adenosine,an Analogue of the Aminoacylation Reaction Intermediate Glutamyl-AMP: Mechanistic and Evolutionary Implications

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNA^Glu or of the non-cognate tRNA^Phe is indicated by the negative values of –TΔS_b, and by the negative value of ΔC_p. On the other hand, the large negative enthalpy is the dominant contribution to ΔG_b in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNA^Phe, but the dissociation constant K _d is decreased 50-fold in the presence of tRNA^Glu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3’-OH oxygen of the 3’-terminal ribose of tRNA^Glu in the Glu-AMS•GluRS•tRNA^Glu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNA^Glu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.     

Ribosome stalling and peptidyl-tRNA drop-off during translational delay at AGA codons

Minigenes encoding the peptide Met–Arg–Arg have been used to study the mechanism of toxicity of AGA codons proximal to the start codon or prior to the termination codon in bacteria. The codon sequences of the ‘mini-ORFs’ employed were initiator, combinations of AGA and CGA, and terminator. Both, AGA and CGA are low-usage Arg codons in ORFs of Escherichia coli but, whilst AGA is translated by the scarce tRNA^Arg4, CGA is recognized by the abundant tRNA^Arg2. Overexpression of minigenes harbouring AGA in the third position, next to a termination codon, was deleterious to the cell and led to the accumulation of peptidyl-tRNA^Arg4 and of the peptidyl-tRNA cognate to the preceding CGA or AGA Arg triplet. The minigenes carrying CGA in the third position were not toxic. Minigene-mediated toxicity and peptidyl-tRNA accumulation were suppressed by overproduction of tRNA^Arg4 but not by overproduction of peptidyl-tRNA hydrolase, an enzyme that is only active on substrates that have been released from the ribosome. Consistent with these findings, peptidyl-tRNA^Arg4 was identified to be mainly associated with ribosomes in a stand-by complex. These and previous results support the hypothesis that the primary mechanism of inhibition of protein synthesis by AGA triplets in pth⁺ cells involves sequestration of tRNAs as peptidyl-tRNA on the stalled ribosome.     

Involvement of glycine transfer ribonucleic acids in development of the posterior silk gland of Bombyx mori

Glycine transfer RNAs from the two physiological phases, V-2, the stage of maximum growth, and V-5, the stage of maximum fibroin production, during the development of the posterior silk gland of Bombyx mori were examined. The tRNAs from both phases could be fractionated into two major isoaccepting species on a benzoylated DEAE-cellulose column. No significant qualitative differences were observed among the tRNAs, but the total amount of the isoaccepting species of tRNA^Gly in each gland of V-5 stage was 6-fold higher than the amount of tRNA^Gly in the V-2 gland. The codon recognition properties of the tRNA^Gly species were examined. It was found that tRNA^Gly₁ responded to the copolymer (G:U) preferentially while tRNA^Gly_II recognized the copolymer (A:G). The ratio between the extent of incorporation of labeled glycine from glycyl-tRNA^Gly₁ and glycyl-tRNA^Gly_II into protein in a cell-free system utilizing polysomes from the V-5 glands was similar to the relative abundance of the isoaccepting species present in the glands at that time. It also reflected the ratio between the corresponding codons assigned for glycine based on the sequence analysis of fibroin-mRNA [Suzuki, Y., and Brown, D. D. (1972) J. Mol. Biol.63: 409]. These results suggest that the abundance of tRNA^Gly in the posterior silk gland and the changes in the relative amounts of the isoaccepting species are quite specific for the development of the gland.     

Sequence-specific recognition of colicin E5, a tRNA-targeting ribonuclease

Colicin E5 is a novel Escherichia coli ribonuclease that specifically cleaves the anticodons of tRNA^Tyr, tRNA^His, tRNA^Asn and tRNA^Asp. Since this activity is confined to its 115 amino acid long C-terminal domain (CRD), the recognition mechanism of E5-CRD is of great interest. The four tRNA substrates share the unique sequence UQU within their anticodon loops, and are cleaved between Q (modified base of G) and 3′ U. Synthetic minihelix RNAs corresponding to the substrate tRNAs were completely susceptible to E5-CRD and were cleaved in the same manner as the authentic tRNAs. The specificity determinant for E5-CRD was YGUN at −1 to +3 of the ‘anticodon’. The YGU is absolutely required and the extent of susceptibility of minihelices depends on N (third letter of the anticodon) in the order A > C > G > U accounting for the order of susceptibility tRNA^Tyr > tRNA^Asp > tRNA^His, tRNA^Asn. Contrastingly, we showed that GpUp is the minimal substrate strictly retaining specificity to E5-CRD. The effect of contiguous nucleotides is inconsistent between the loop and linear RNAs, suggesting that nucleotide extension on each side of GpUp introduces a structural constraint, which is reduced by a specific loop structure formation that includes a 5′ pyrimidine and 3′ A.     

Position 34 of tRNA is a discriminative element for m5C38 modification by human DNMT2

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 tRNA^Asp(GUC), tRNA^Gly(GCC) and tRNA^Val(AAC). Intriguingly, for tRNA^Asp(GUC) and tRNA^Gly(GCC), G34 is the discriminator element; whereas for tRNA^Val(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.     

Chloroplastic methionyl-tRNA synthetase from wheat

Wheat chloroplastic methionyl-tRNA synthetase was isolated and appeared to be a monomer with a molecular weight of 75,000 daltons. Its catalytical properties in the aminoacylation for various isoacceptors tRNA_s^Met from E. coli and wheat germ revealed a recognition of prokaryotic tRNAs and wheat cytoplasmic tRNA_i^Met, but not tRNA_m^Met. Using pI determinations and catalytical properties, it could be detected in non-chloroplastic quiescent wheat germ a form of methionyl-tRNA synthetase having the same properties as the chloroplastic one's.     

The crystal structure of yeast mitochondrial ThrRS in complex with the canonical threonine tRNA

In mitochondria of Saccharomyces cerevisiae, a single aminoacyl-tRNA synthetase (aaRS), MST1, aminoacylates two isoacceptor tRNAs, tRNA₁^Thr and tRNA₂^Thr, that harbor anticodon loops of different size and sequence. As a result of this promiscuity, reassignment of the CUN codon box from leucine to threonine is facilitated. However, the mechanism by which a single aaRS binds distinct anticodon loops with high specificity is not well understood. Herein, we present the crystal structure of MST1 in complex with the canonical tRNA₂^Thr and non-hydrolyzable analog of threonyl adenylate. Our structure reveals that the dimeric arrangement of MST1 is essential for binding the 5′-phosphate, the second base pair of the acceptor stem, the first two base pairs of the anticodon stem and the first nucleotide of the variable arm. Further, in contrast to the bacterial ortholog that ‘reads’ the entire anticodon sequence, MST1 recognizes bases in the second and third position and the nucleotide upstream of the anticodon sequence. We speculate that a flexible loop linking strands β4 and β5 may be allosteric regulator that establishes cross-subunit communication between the aminoacylation and tRNA-binding sites. We also propose that structural features of the anticodon-binding domain in MST1 permit binding of the enlarged anticodon loop of tRNA₁^Thr.     

EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways

The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and P_i release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.