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 tRNAAsp and tRNAAsn generating a correctly charged Asp-tRNAAsp and an erroneous Asp-tRNAAsn . 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 tRNAAsp over tRNAAsn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNAAsp and was unable to aspartylate tRNAAsn . 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.

Bacterial Aspartyl-tRNA Synthetase Has Glutamyl-tRNA Synthetase Activity.

The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNAAsn via Asp-tRNAAsn. This intermediate is produced by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) that has retained its ability to also generate Asp-tRNAAsp. Here we report the discovery that ND-AspRS and its discriminating counterpart, AspRS, are also capable of specifically producing Glu-tRNAGlu, without producing misacylated tRNAs like Glu-tRNAAsn, Glu-tRNAAsp, or Asp-tRNAGlu, thus maintaining the fidelity of the genetic code. Consequently, bacterial AspRSs have glutamyl-tRNA synthetase-like activity that does not contaminate the proteome via amino acid misincorporation.

Crystal structure of the N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori.

The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNAAsp and tRNAAsn. Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS1-104) from the human-pathogenic bacterium Helicobacter pylori is reported at 2.0 Šresolution. The apo form of H. pylori ND-AspRS1-104 shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) from Escherichia coli and ND-AspRS from Pseudomonas aeruginosa, allowing recognition elements to be proposed for tRNAAsp and tRNAAsn. It is proposed that a long loop (Arg77-Lys90) in this H. pylori domain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS from E. coli and ND-AspRS from P. aeruginosa suggests that turns E and F (78GAGL81 and 83NPKL86) in H. pylori ND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNAAsp (34GUC36) and tRNAAsn (34GUU36). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.

The predatory bacterium Bdellovibrio bacteriovorus aspartyl-tRNA synthetase recognizes tRNAAsn as a substrate.

The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNAAsn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNAAsn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.

Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis.

The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNA(Asn). The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNA(Asn). S. aureus thus encodes both the direct and indirect routes for Asn-tRNA(Asn) formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase.

Two-codon T-box riboswitch binding two tRNAs.

T-box riboswitches control transcription of downstream genes through the tRNA-binding formation of terminator or antiterminator structures. Previously reported T-boxes were described as single-specificity riboswitches that can bind specific tRNA anticodons through codon-anticodon interactions with the nucleotide triplet of their specifier loop (SL). However, the possibility that T-boxes might exhibit specificity beyond a single tRNA had been overlooked. In Clostridium acetobutylicum, the T-box that regulates the operon for the essential tRNA-dependent transamidation pathway harbors a SL with two potential overlapping codon positions for tRNA(Asn) and tRNA(Glu). To test its specificity, we performed extensive mutagenic, biochemical, and chemical probing analyses. Surprisingly, both tRNAs can efficiently bind the SL in vitro and in vivo. The dual specificity of the T-box is allowed by a single base shift on the SL from one overlapping codon to the next. This feature allows the riboswitch to sense two tRNAs and balance the biosynthesis of two amino acids. Detailed genomic comparisons support our observations and suggest that "flexible" T-box riboswitches are widespread among bacteria, and, moreover, their specificity is dictated by the metabolic interconnection of the pathways under control. Taken together, our results support the notion of a genome-dependent codon ambiguity of the SLs. Furthermore, the existence of two overlapping codons imposes a unique example of tRNA-dependent regulation at the transcriptional level.

Mitochondrial myopathy in a child with a muscle-restricted mutation in the mitochondrial transfer RNAAsn gene.

We report an 11-year-old boy with exercise-related myopathy, and a novel mutation m.5669G>A in the mitochondrial tRNA Asparagine gene (mt-tRNA(Asn), MTTN). Muscle biopsy studies showed COX-negative, SDH-positive fibers at histochemistry and biochemical defects of oxidative metabolism. The m.5669G>A mutation was present only in patient's muscle resulting in the first muscle-specific MTTN mutation. Mt-tRNA(Asn) steady-state levels and in silico predictions supported the pathogenicity of this mutation. A mitochondrial myopathy should be considered in the differential diagnosis of exercise intolerance in children.

Two distinct regions in Staphylococcus aureus GatCAB guarantee accurate tRNA recognition.

In many prokaryotes the biosynthesis of the amide aminoacyl-tRNAs, Gln-tRNA(Gln) and Asn-tRNA(Asn), proceeds by an indirect route in which mischarged Glu-tRNA(Gln) or Asp-tRNA(Asn) is amidated to the correct aminoacyl-tRNA catalyzed by a tRNA-dependent amidotransferase (AdT). Two types of AdTs exist: bacteria, archaea and organelles possess heterotrimeric GatCAB, while heterodimeric GatDE occurs exclusively in archaea. Bacterial GatCAB and GatDE recognize the first base pair of the acceptor stem and the D-loop of their tRNA substrates, while archaeal GatCAB recognizes the tertiary core of the tRNA, but not the first base pair. Here, we present the crystal structure of the full-length Staphylococcus aureus GatCAB. Its GatB tail domain possesses a conserved Lys rich motif that is situated close to the variable loop in a GatCAB:tRNA(Gln) docking model. This motif is also conserved in the tail domain of archaeal GatCAB, suggesting this basic region may recognize the tRNA variable loop to discriminate Asp-tRNA(Asn) from Asp-tRNA(Asp) in archaea. Furthermore, we identified a 3(10) turn in GatB that permits the bacterial GatCAB to distinguish a U1-A72 base pair from a G1-C72 pair; the absence of this element in archaeal GatCAB enables the latter enzyme to recognize aminoacyl-tRNAs with G1-C72 base pairs.

Low modularity of aminoacyl-tRNA substrates in polymerization by the ribosome.

Aminoacyl-transfer RNAs contain four standardized units: amino acids, an invariant 3'-terminal CCA, trinucleotide anticodons and tRNA bodies. The degree of interchangeability of the three variable modules is poorly understood, despite its role in evolution and the engineering of translation to incorporate unnatural amino acids. Here, a purified translation system is used to investigate effects of various module swaps on the efficiency of multiple ribosomal incorporations of unnatural aminoacyl-tRNA substrates per peptide product. The yields of products containing three to five adjacent l-amino acids with unnatural side chains are low and cannot be improved by optimization or explained simply by any single factor tested. Though combinations of modules that allow quantitative single unnatural incorporations are found readily, finding combinations that enable efficient synthesis of products containing multiple unnatural amino acids is challenging. This implies that assaying multiple, as opposed to single, incorporations per product is a more stringent assay of substrate activity. The unpredictability of most results illustrates the multifactorial nature of substrate recognition and the value of synthetic biology for testing our understanding of translation. Data indicate that the degree of interchangeability of the modules of aminoacyl-tRNAs is low.

Ribosomal frameshifting in response to hypomodified tRNAs in Xenopus oocytes.

We used Xenopus oocytes as an intracellular system to study ribosomal frameshifting. Microinjection of oocytes with a construct encoding the naturally occurring UUU or AAC codon at the frameshift site demonstrated that the level of frameshifting was similar or lower than found normally in retroviral frameshifting in mammalian cells, suggesting that oocytes are a reliable system to study this event. Phenylalanine (Phe) or asparagine (Asn) tRNAs with and without the highly modified wyebutoxine (Y) or queuosine (Q) base, respectively, were microinjected to assess their ability to promote frameshifting. tRNAPhe+Y inhibited the level of frameshifting, while tRNAPhe-Y promoted frameshifting providing evidence that the hypomodified form does not act only to enhance frameshifting, but is an essential requirement. Both tRNAAsn+Q and tRNAAsn-Q were used indiscriminately in frameshifting, whether the frameshift site contained the wild-type AAC, or the mutant AAU codon, suggesting that Q base modification status does not influence this process.

Conserved discrimination against misacylated tRNAs by two mesophilic elongation factor Tu orthologs.

Elongation factor Tu (EF-Tu) binds and loads elongating aminoacyl-tRNAs (aa-tRNAs) onto the ribosome for protein biosynthesis. Many bacteria biosynthesize Gln-tRNA (Gln) and Asn-tRNA (Asn) by an indirect, two-step pathway that relies on the misacylated tRNAs Glu-tRNA (Gln) and Asp-tRNA (Asn) as intermediates. Previous thermodynamic and experimental analyses have demonstrated that Thermus thermophilus EF-Tu does not bind Asp-tRNA (Asn) and predicted a similar discriminatory response against Glu-tRNA (Gln) [Asahara, H., and Uhlenbeck, O. (2005) Biochemistry 46, 6194-6200; Roy, H., et al. (2007) Nucleic Acids Res. 35, 3420-3430]. By discriminating against these misacylated tRNAS, EF-Tu plays a direct role in preventing misincorporation of aspartate and glutamate into proteins at asparagine and glutamine codons. Here we report the characterization of two different mesophilic EF-Tu orthologs, one from Escherichia coli, a bacterium that does not utilize either Glu-tRNA (Gln) or Asp-tRNA (Asn), and the second from Helicobacter pylori, an organism in which both misacylated tRNAs are essential. Both EF-Tu orthologs discriminate against these misacylated tRNAs, confirming the prediction that Glu-tRNA (Gln), like Asp-tRNA (Asn), will not form a complex with EF-Tu. These results also demonstrate that the capacity of EF-Tu to discriminate against both of these aminoacyl-tRNAs is conserved even in bacteria like E. coli that do not generate either misacylated tRNA.

The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis.

Asparagine, one of the 22 genetically encoded amino acids, can be synthesized by a tRNA-dependent mechanism. So far, this type of pathway was believed to proceed via two independent steps. A nondiscriminating aspartyl-tRNA synthetase (ND-DRS) first generates a mischarged aspartyl-tRNAAsn that dissociates from the enzyme and binds to a tRNA-dependent amidotransferase (AdT), which then converts the tRNA-bound aspartate into asparagine. We show herein that the ND-DRS, tRNAAsn, and AdT assemble into a specific ribonucleoprotein complex called transamidosome that remains stable during the overall catalytic process. Our results indicate that the tRNAAsn-mediated linkage between the ND-DRS and AdT enables channeling of the mischarged aspartyl-tRNAAsn intermediate between DRS and AdT active sites to prevent challenging of the genetic code integrity. We propose that formation of a ribonucleoprotein is a general feature for tRNA-dependent amino acid biosynthetic pathways that are remnants of earlier stages when amino acid synthesis and tRNA aminoacylation were coupled.

Novel tRNA aminoacylation mechanisms.

In nature, ribosomally synthesized proteins can contain at least 22 different amino acids: the 20 common amino acids as well as selenocysteine and pyrrolysine. Each of these amino acids is inserted into proteins codon-specifically via an aminoacyl-transfer RNA (aa-tRNA). In most cases, these aa-tRNAs are biosynthesized directly by a set of highly specific and accurate aminoacyl-tRNA synthetases (aaRSs). However, in some cases aaRSs with relaxed or novel substrate specificities cooperate with other enzymes to generate specific canonical and non-canonical aminoacyl-tRNAs.

Structural elements defining elongation factor Tu mediated suppression of codon ambiguity.

In most prokaryotes Asn-tRNA(Asn) and Gln-tRNA(Gln) are formed by amidation of aspartate and glutamate mischarged onto tRNA(Asn) and tRNA(Gln), respectively. Coexistence in the organism of mischarged Asp-tRNA(Asn) and Glu-tRNA(Gln) and the homologous Asn-tRNA(Asn) and Gln-tRNA(Gln) does not, however, lead to erroneous incorporation of Asp and Glu into proteins, since EF-Tu discriminates the misacylated tRNAs from the correctly charged ones. This property contrasts with the canonical function of EF-Tu, which is to non-specifically bind the homologous aa-tRNAs, as well as heterologous species formed in vitro by aminoacylation of non-cognate tRNAs. In Thermus thermophilus that forms the Asp-tRNA(Asn) intermediate by the indirect pathway of tRNA asparaginylation, EF-Tu must discriminate the mischarged aminoacyl-tRNAs (aa-tRNA). We show that two base pairs in the tRNA T-arm and a single residue in the amino acid binding pocket of EF-Tu promote discrimination of Asp-tRNA(Asn) from Asn-tRNA(Asn) and Asp-tRNA(Asp) by the protein. Our analysis suggests that these structural elements might also contribute to rejection of other mischarged aa-tRNAs formed in vivo that are not involved in peptide elongation. Additionally, these structural features might be involved in maintaining a delicate balance of weak and strong binding affinities between EF-Tu and the amino acid and tRNA moieties of other elongator aa-tRNAs.

The Helicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNAGln.

The amide aminoacyl-tRNAs, Gln-tRNA(Gln) and Asn-tRNA(Asn), are formed in many bacteria by a pretranslational tRNA-dependent amidation of the mischarged tRNA species, Glu-tRNA(Gln) or Asp-tRNA(Asn). This conversion is catalyzed by a heterotrimeric amidotransferase GatCAB in the presence of ATP and an amide donor (Gln or Asn). Helicobacter pylori has a single GatCAB enzyme required in vivo for both Gln-tRNA(Gln) and Asn-tRNA(Asn) synthesis. In vitro characterization reveals that the enzyme transamidates Asp-tRNA(Asn) and Glu-tRNA(Gln) with similar efficiency (k(cat)/K(m) of 1368.4 s(-1)/mM and 3059.3 s(-1)/mM respectively). The essential glutaminase activity of the enzyme is a property of the A-subunit, which displays the characteristic amidase signature sequence. Mutations of the GatA catalytic triad residues (Lys(52), Ser(128), Ser(152)) abolished glutaminase activity and consequently the amidotransferase activity with glutamine as the amide donor. However, the latter activity was rescued when the mutant enzymes were presented with ammonium chloride. The presence of Asp-tRNA(Asn) and ATP enhances the glutaminase activity about 22-fold. H. pylori GatCAB uses the amide donor glutamine 129-fold more efficiently than asparagine, suggesting that GatCAB is a glutamine-dependent amidotransferase much like the unrelated asparagine synthetase B. Genomic analysis suggests that most bacteria synthesize asparagine in a glutamine-dependent manner, either by a tRNA-dependent or in a tRNA-independent route. However, all known bacteria that contain asparagine synthetase A form Asn-tRNA(Asn) by direct acylation catalyzed by asparaginyl-tRNA synthetase. Therefore, bacterial amide aminoacyl-tRNA formation is intimately tied to amide amino acid metabolism.

Inhibition by L-aspartol adenylate of a nondiscriminating aspartyl-tRNA synthetase reveals differences between the interactions of its active site with tRNA(Asp) and tRNA(Asn).

Asparaginyl-tRNA formation in Pseudomonas aeruginosa PAO1 involves a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) which forms Asp-tRNA(Asp) and Asp-tRNA(Asn), and a tRNA-dependent amidotransferase which transamidates the latter into Asn-tRNA(Asn). We report here that the inhibition of this ND-AspRS by L-aspartol adenylate (Asp-ol-AMP), a stable analog of the natural reaction intermediate L-aspartyl adenylate, is biphasic because the aspartylation of the two tRNA substrates of ND-AspRS, tRNA(Asp) and tRNA(Asn), are inhibited with different Ki values (41 microM and 215 microM, respectively). These results reveal that the two tRNA substrates of ND-AspRS interact differently with its active site. Yeast tRNA(Asp) transcripts with some identity elements replaced by those of tRNA(Asn) have their aspartylation inhibited with Ki values different from that for the wild-type transcript. Therefore, aminoacyl adenylate analogs, which are competitive inhibitors of their cognate aminoacyl-tRNA synthetase, can be used to probe rapidly the role of various structural elements in positioning the tRNA acceptor end in the active site.

Co-evolution of the archaeal tRNA-dependent amidotransferase GatCAB with tRNA(Asn).

The important identity elements in tRNA(Gln) and tRNA(Asn) for bacterial GatCAB and in tRNA(Gln) for archaeal GatDE are the D-loop and the first base pair of the acceptor stem. Here we show that Methanothermobacter thermautotrophicus GatCAB, the archaeal enzyme, is different as it discriminates Asp-tRNA(Asp) and Asp-tRNA(Asn) by use of U49, the D-loop and to a lesser extent the variable loop. Since archaea possess the tRNA(Gln)-specific amidotransferase GatDE, the archaeal GatCAB enzyme evolved to recognize different elements in tRNA(Asn) than those recognized by GatDE or by the bacterial GatCAB enzyme in their tRNA substrates.

A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine.

In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNAAsn and tRNAGln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNAAsn and Gln-tRNAGln. So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNAAsn and tRNAAsp variants, that amidation of Asp acylating tRNAAsn is promoted by the base pair U1-A72 whereas the G1-C72 pair and presence of the supernumerary nucleotide U20A in the D-loop of tRNAAsp prevent amidation. We predict, based on comparison of tRNAGln and tRNAGlu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNAGln, that the same combination of nucleotides also rules specific tRNA-dependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G46 and U47 of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.

Determination of queuosine derivatives by reverse-phase liquid chromatography for the hypomodification study of Q-bearing tRNAs from various mammal liver cells.

Three queuosine derivatives (Q-derivatives) have been found at position 34 of four mammalian so-called Q-tRNAs: queuosine (Q) in tRNA(Asn) and tRNA(His), mannosyl-queuosine (manQ) in tRNA(Asp), and galactosyl-queuosine (galQ) in tRNA(Tyr). An analytical procedure based on the combined means of purified tRNA isolation from liver cells and ribonucleoside analysis by reverse-phase high performance liquid chromatography coupled with real-time UV-spectrometry (RPLC-UV) was developed for the quantitative analysis of the three Q-derivatives present in total tRNA from liver tissues and liver cell cultures. Using this analytical procedure, the rates of Q-tRNA modification were studied in total tRNAs from various mammalian hepatic cells. Our results show that the four Q-tRNAs are fully modified in liver tissues from adult mammals, regardless of the mammal species. However, a lack in the Q-modification level was observed in Q-tRNAs from newborn rat liver, as well in Q-tRNAs from normal rat liver cell cultures growing in a low queuine content medium, and from a rat hepatoma cell line. It is noteworthy that in all cases of Q-tRNA hypomodification, our analytical procedure showed that tRNA(Asp) is always the least affected by the hypomodification. The biological significance of this phenomenon is discussed.

RSEARCH: finding homologs of single structured RNA sequences.

BACKGROUND: For many RNA molecules, secondary structure rather than primary sequence is the evolutionarily conserved feature. No programs have yet been published that allow searching a sequence database for homologs of a single RNA molecule on the basis of secondary structure. RESULTS: We have developed a program, RSEARCH, that takes a single RNA sequence with its secondary structure and utilizes a local alignment algorithm to search a database for homologous RNAs. For this purpose, we have developed a series of base pair and single nucleotide substitution matrices for RNA sequences called RIBOSUM matrices. RSEARCH reports the statistical confidence for each hit as well as the structural alignment of the hit. We show several examples in which RSEARCH outperforms the primary sequence search programs BLAST and SSEARCH. The primary drawback of the program is that it is slow. The C code for RSEARCH is freely available from our lab's website. CONCLUSION: RSEARCH outperforms primary sequence programs in finding homologs of structured RNA sequences.