Crystal structure of glutamyl-queuosine tRNAAsp synthetase complexed with L-glutamate: structural elements mediating tRNA-independent activation of glutamate and glutamylation of tRNAAsp anticodon

Glutamyl-queuosine tRNA^Asp synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNA^Asp. Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNA^Glu, Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3′ accepting end of the cognate tRNA^Glu, Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNA^Asp. In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS·Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNA^Asp among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNA^Asp that determine recognition by Glu-Q-RS. The analyses made on tRNA^Asp and tRNA^Asn show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.     

Queuine in plants and plant tRNAs: Differences between embryonic tissue and mature leaves

In eubacterial and eukaryotic tRNAs specific for Asn, Asp, His and Tyr the modified deazaguanosinederivative queuosine occurs in position 34, the first position of the anticodon. Analysis of unfractionated tRNAs from wheat and from tobacco leaves shows that these tRNAs contain high amounts of guanosine (G) in place of queuosine (Q). This was measured by the exchange of G34 for [³H]guanine catalysed by the specific tRNA guanine transglycosylase from E. coli. Upon gel electrophoretic separation of the labeled tRNAs, seven Q-deficient tRNA species including isoacceptors are detectable. Two are identified as cytoplasmic tRNAs^Tyr and tRNA^Asp and two represent chloroplast tRNA^Tyr isoacceptors. In contrast to leaf cytoplasm and chloroplasts, wheat germ has low amounts of tRNAs with G34 in place of Q.A new enzymatic assay is described for quantitation of free queuine in cells and tissues. Analysis of queuine in plant tissues shows that wheat germ contains about 200 ng queuine per g wet weight. In wheat and tobacco leaves queuine is present, if at all, in amounts lower than 10 ng/g wet weight. The absence of Q in tRNAs from plant leaves is therefore caused by a deficiency of queuine. Tobacco cells cultivated in a synthetic medium without added queuine do not contain Q in tRNA, indicating that these rapidly growing cells do not synthesize queuine de novo.     

The structure of yeast tRNAAsp. A model for tRNA interacting with messenger RNA

Abstract

The anticodon of yeast tRNA^Asp, GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNA^Asp molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNA^Phe. In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNA^Asp T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNA^Phe. This variation is a consequence of the anticodon-anticodon base pairing which rigidities the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNA^Asp substantiate such a correlation.     

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.     

Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine

Dnmt2 enzymes are cytosine-5 methyltransferases that methylate C38 of several tRNAs. We report here that the activities of two Dnmt2 homologs, Pmt1 from Schizosaccharomyces pombe and DnmA from Dictyostelium discoideum, are strongly stimulated by prior queuosine (Q) modification of the substrate tRNA. In vivo tRNA methylation levels were stimulated by growth of cells in queuine-containing medium; in vitro Pmt1 activity was enhanced on Q-containing RNA; and queuine-stimulated in vivo methylation was abrogated by the absence of the enzyme that inserts queuine into tRNA, eukaryotic tRNA-guanine transglycosylase. Global analysis of tRNA methylation in S. pombe showed a striking selectivity of Pmt1 for tRNA^Asp methylation, which distinguishes Pmt1 from other Dnmt2 homologs. The present analysis also revealed a novel Pmt1- and Q-independent tRNA methylation site in S. pombe, C34 of tRNA^Pro. Notably, queuine is a micronutrient that is scavenged by higher eukaryotes from the diet and gut microflora. This work therefore reveals an unanticipated route by which the environment can modulate tRNA modification in an organism.     

Anticodon-binding domain swapping in a nondiscriminating aspartyl-tRNA synthetase reveals contributions to tRNA specificity and catalytic activity

The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNA^Asp and tRNA^Asn generating a correctly charged Asp-tRNA^Asp and an erroneous Asp-tRNA^Asn. This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNA^Asp over tRNA^Asn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNA^Asp and was unable to aspartylate tRNA^Asn. The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity.     

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.     

Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.     

Structural insights into translational recoding by frameshift suppressor tRNASufJ

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5′ or 3′ direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNA^SufJ, a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNA^SufJ contains an insertion 5′ to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASL^SufJ or tRNA^SufJ does not affect its affinity for the A site of the ribosome. Structural analyses of both ASL^SufJ and ASL^Thr bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34–37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASL^SufJ imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNA^SufJ during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting.     

Association of tRNA acceptor identity with phosphate-sugar backbone interactions observed in the crystal structure of the Escherichia coli glutaminyl-tRNA synthetase-tRNA complex

We isolated several mutants with nucleotide substitutions in alanine tRNA (tRNA^Ala) that resulted in glutamine tRNA (tRNA^Gli) acceptor identity in Escherichia coli. These substitutions were in three regions of tRNA structure not previously associated with tRNA^Gln acceptor identity. Only the phosphate-sugar backbone moieties of these nucleotides interact with the enzyme in the previously determined X-ray crystal structure of the complex between tRNA^Gln and glutaminyl-tRNA synthetase. We conclude that these sequence-dependent phosphate-sugar backbone interactions contribute to tRNA^Gln identity, and argue that the interactions help communicate enzyme recognition of the anticodon to the acceptor end of the tRNA and the catalytic center of the enzyme.     

Purification and nucleoside composition of a Q* - containing aspartic acid tRNA from Drosophila.

One form of aspartic acid tRNA from $\text{Drosophila}$,$\text{melanogaster}$ (tRNA_2δ^Asp) is selectively bound to columns of Con A-Sepharose. Unlike the other Q-containing tRNAs of $\text{Drosophila}$, it therefore appears that tRNA^Asp contains the more highly modified nucleoside, Q¹ (mannose form) in its anticodon. This is further supported by the chromatographic insensitivity of tRNA_2δ^Asp to NaIO₄ treatment. Utilizing Con A-Sepharose chromatography, tRNA_2δ^Asp from $\text{Drosophila}$ was purified and its nucleoside composition determined by chemical tritium labelling. In addition to the major nucleosides, this tRNA contains rT, hU, m⁵C, ψ, and Q¹, but no other modified nucleosides. Its nucleoside composition is very similar to yeast tRNA^Asp.     

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.     

Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment

Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNA^Opt. We suspected a modification of the tRNA^Opt_AUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNA^Opt_AUG is converted to inosine. We identified tRNA^Opt_AUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms.     

A critical role of water in the specific cleavage of the anticodon loop of some eukaryotic methionine initiator tRNAs

We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond. A literature search indicated that there is also the case for human initiator tRNA^Met but not for yeast tRNA^Met _i or E. coli tRNA^Met _f. All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop. It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNA^Met _i susceptible to the specific cleavage reaction. Using crystallographic data of tRNA^Met _f of E. coli with U33, we modeled the anticodon loop of this tRNA with C33. We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond. We conclude that a single nucleotide change in the primary structure of tRNA^Met _i made changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.