tRNA recognition by tRNA-guanine transglycosylase from Escherichia coli: the role of U33 in U-G-U sequence recognition.

In eubacteria, the biosynthesis of queuine, a modified base found in the wobble position (#34) of tRNAs coding for Tyr, His, Asp, and Asn, occurs via a multistep pathway. One of the key enzymes in this pathway, tRNA-guanine transglycosylase (TGT), exchanges the genetically encoded guanine at position 34 with a queuine precursor, preQ1. Previous studies have identified a minimal positive RNA recognition motif for Escherichia coli TGT consisting of a stable minihelix that contains a U-G-U sequence starting at the second position of its seven base anticodon loop. Recently, we reported that TGT was capable of recognizing the U-G-U sequence outside of this limited structural context. To further characterize the ability of TGT to recognize the U-G-U sequence in alternate contexts, we constructed mutants of the previously characterized E. coli tRNA(Tyr) minihelix. The U-G-U sequence was shifted to various positions within the anticodon loop of these mutants. Characterization of these analogs demonstrates that in addition to the normal U33G34U35 position, TGT can also recognize the U34G35U36 analog (UGU(+1)). The other analogs were not active. This indicates that the recognition of the U-G-U sequence is not strictly dependent upon its position relative to the stem. In E. coli, the full-length tRNA with a U34G35U36 anticodon sequence is one of the isoacceptors that codes for threonine. We found that TGT is able to recognize tRNA(Thr(UGU)) but only in the absence of a uridine at position 33. U33, an invariant base present in all tRNAs, has been shown to strongly influence the conformation of the anticodon loop of certain tRNAs. We find that mutation of this base confers on TGT the ability to recognize U34G35U36, and suggests that loop conformation affects recognition. The fact that the other analogs were not active indicates that although TGT is capable of recognizing the U-G-U sequence in additional contexts, this recognition is not indiscriminate.     

The Escherichia coli tRNA-guanine transglycosylase can recognize and modify DNA.

tRNA-guanine transglycosylase (TGT) catalyzes the exchange of queuine (or a precursor) for guanine 34 in tRNA. The minimal RNA recognition motif for TGT has been found to involve a UGU sequence in the anticodon loop of the queuine-cognate tRNAs. Recent studies have shown that the enzyme is capable of recognizing the UGU sequence in alternative contexts (Kung, F. L., Nonekowski, S., and Garcia, G. A. (2000) RNA 6, 233-244) and have investigated the role of the first U of the UGU sequence in tRNA recognition by TGT (Nonekowski, S. T., and Garcia, G. A. (2001) RNA 7, 1432-1441). The TGT reaction involves the breakage and re-formation of a glycosidic bond. To rule out a potential chemical mechanism involving the 2'-hydroxyl at position 34, we synthesized and evaluated an RNA minihelix with 2'-deoxy-G at 34. The high level of activity exhibited by this analogue indicates that the 2'-hydroxyl of G(34) is not required for catalysis. Furthermore, we find that TGT can recognize analogues composed entirely of DNA, but only when 2'-deoxyuridines replace the thymidines in the DNA. The requirement for uridine bases for recognition is perhaps not surprising given the UGU recognition motif for TGT. However, it is not clear if the uracil requirement is due to specific recognition by TGT or due to the effect of uracils on the conformation of the oligonucleotide.     

Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate

Prokaryotic tRNA guanine transglycosylase (TGT) catalyzes replacement of guanine (G) by 7-aminomethyl-7-deazaguanine (PreQ1) at the wobble position of four specific tRNAs. Addition of 9-deazaguanine (9dzG) to a reaction mixture of Zymomonas mobilis TGT and an RNA substrate allowed us to trap, purify and crystallize a chemically competent covalent intermediate of the TGT-catalyzed reaction. The crystal structure of the TGT-RNA-9dzG ternary complex at a resolution of 2.9 A reveals, unexpectedly, that RNA is tethered to TGT through the side chain of Asp280. Thus, Asp280, instead of the previously proposed Asp102, acts as the nucleophile for the reaction. The RNA substrate adopts an unusual conformation, with four out of seven nucleotides in the loop region flipped out. Interactions between TGT and RNA revealed by the structure provide the molecular basis of the RNA substrate requirements by TGT. Furthermore, reaction of PreQ1 with the crystallized covalent intermediate provides insight into the necessary structural changes required for the TGT-catalyzed reaction to occur.     

Efficient aminoacylation of tRNA(Lys,3) by human lysyl-tRNA synthetase is dependent on covalent continuity between the acceptor stem and the anticodon domain

In this work, we probe the role of the anticodon in tRNA recognition by human lysyl-tRNA synthetase (hLysRS). Large decreases in aminoacylation efficiency are observed upon mutagenesis of anticodon positions U35 and U36 of human tRNA(Lys,3). A minihelix derived from the acceptor-TPsiC stem-loop domain of human tRNA(Lys,3)was not specifically aminoacylated by the human enzyme. The presence of an anticodon-derived stem-loop failed to stimulate aminoacylation of the minihelix. Thus, covalent continuity between the acceptor stem and anticodon domains appears to be an important requirement for efficient charging by hLysRS. To further examine the mechanism of communication between the critical anticodon recognition elements and the catalytic site, a two piece semi-synthetic tRNA(Lys, 3)construct was used. The wild-type semi-synthetic tRNA contained a break in the phosphodiester backbone in the D loop and was an efficient substrate for hLysRS. In contrast, a truncated variant that lacked nucleotides 8-17 in the D stem-loop displayedseverely reduced catalytic efficiency. The elimination of key tRNA tertiary structural elements has little effect on anticodon-dependent substrate binding but severely impacts formation of the proper transition state for catalysis. Taken together, our studies provide new insights into human tRNA structural requirements for effective transmission of the anticodon recognition signal to the distal acceptor stem domain.     

The RNA-binding PUA domain of archaeal tRNA-guanine transglycosylase is not required for archaeosine formation

Bacterial tRNA-guanine transglycosylase (TGT) replaces the G in position 34 of tRNA with preQ(1), the precursor to the modified nucleoside queuosine. Archaeal TGT, in contrast, substitutes preQ(0) for the G in position 15 of tRNA as the first step in archaeosine formation. The archaeal enzyme is about 60% larger than the bacterial protein; a carboxyl-terminal extension of 230 amino acids contains the PUA domain known to contact the four 3'-terminal nucleotides of tRNA. Here we show that the C-terminal extension of the enzyme is not required for the selection of G15 as the site of base exchange; truncated forms of Pyrococcus furiosus TGT retain their specificity for guanine exchange at position 15. Deletion of the PUA domain causes a 4-fold drop in the observed k(cat) (2.8 x 10(-3) s(-1)) and results in a 75-fold increased K(m) for tRNA(Asp)(1.2 x 10(-5) m) compared with full-length TGT. Mutations in tRNA(Asp) altering or abolishing interactions with the PUA domain can compete with wild-type tRNA(Asp) for binding to full-length and truncated TGT enzymes. Whereas the C-terminal domains do not appear to play a role in selection of the modification site, their relevance for enzyme function and their role in vivo remains to be discovered.     

Crystal structure of archaeosine tRNA-guanine transglycosylase

Archaeosine tRNA-guanine transglycosylase (ArcTGT) catalyzes the exchange of guanine at position 15 in the D-loop of archaeal tRNAs with a free 7-cyano-7-deazaguanine (preQ(0)) base, as the first step in the biosynthesis of an archaea-specific modified base, archaeosine (7-formamidino-7-deazaguanosine). We determined the crystal structures of ArcTGT from Pyrococcus horikoshii at 2.2 A resolution and its complexes with guanine and preQ(0), at 2.3 and 2.5 A resolutions, respectively. The N-terminal catalytic domain folds into an (alpha/beta)(8) barrel with a characteristic zinc-binding site, showing structural similarity with that of the bacterial queuosine TGT (QueTGT), which is involved in queuosine (7-[[(4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]methyl]-7-deazaguanosine) biosynthesis and targets the tRNA anticodon. ArcTGT forms a dimer, involving the zinc-binding site and the ArcTGT-specific C-terminal domain. The C-terminal domains have novel folds, including an OB fold-like "PUA domain", whose sequence is widely conserved in eukaryotic and archaeal RNA modification enzymes. Therefore, the C-terminal domains may be involved in tRNA recognition. In the free-form structure of ArcTGT, an alpha-helix located at the rim of the (alpha/beta)(8) barrel structure is completely disordered, while it is ordered in the guanine-bound and preQ(0)-bound forms. Structural comparison of the ArcTGT.preQ(0), ArcTGT.guanine, and QueTGT.preQ(1) complexes provides novel insights into the substrate recognition mechanisms of ArcTGT.     

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: essential elements for recognition of tRNA substrates within the anticodon stem-loop

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the alkylation of the exocyclic amine of A37 by a dimethylallyl unit in tRNAs with an adenosine in the third anticodon position (position 36). By use of purified recombinant enzyme, steady- state kinetic studies were conducted with chemically synthesized RNA oligoribonucleotides to determine the essential elements within the tRNA anticodon stem-loop structure required for recognition by the enzyme. A 17-base oligoribonucleotide corresponding to the anticodon stem-loop of E. coli tRNA(Phe) formed a stem-loop minihelix (minihelix(Phe)) when annealed rapidly on ice, while the same molecule formed a duplex structure with a central loop when annealed slowly at higher concentrations. Both the minihelix and duplex structures gave k(cat)s similar to that for the normal substrate (full-length tRNA(Phe) unmodified at A37), although the K(m) for minihelix(Phe) was approximately 180-fold higher than full-length tRNA. The A36-A37-A38 motif, which is completely conserved in tRNAs modified by the enzyme, was found to be important for modification. Changing A36 to G in the minihelix resulted in a 260-fold reduction in k(cat) compared to minihelix(Phe) and a 13-fold increase in K(m). An A38G variant was modified with a 9-fold reduction in k(cat) and a 5-fold increase in K(m). A random coil 17-base oligoribonucleotide in which the loop sequence of E. coli tRNA(Phe) was preserved, but the 5 base pair helix stem was completely disrupted and showed no measurable activity, indicating that a helix-loop structure is essential for recognition. Finally, altering the identity of several base pairs in the helical stem did not have a major effect on catalytic efficiency, suggesting that the enzyme does not make base-specific contacts important for binding or catalysis in this region.     

Kluyveromyces lactis gamma-toxin, a ribonuclease that recognizes the anticodon stem loop of tRNA

Kluyveromyces lactis gamma-toxin is a tRNA endonuclease that cleaves Saccharomyces cerevisiae [see text] between position 34 and position 35. All three substrate tRNAs carry a 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) residue at position 34 (wobble position) of which the mcm(5) group is required for efficient cleavage. However, the different cleavage efficiencies of mcm(5)s(2)U(34)-containing tRNAs suggest that additional features of these tRNAs affect cleavage. In the present study, we show that a stable anticodon stem and the anticodon loop are the minimal requirements for cleavage by gamma-toxin. A synthetic minihelix RNA corresponding to the anticodon stem loop (ASL) of the natural substrate [see text] is cleaved at the same position as the natural substrate. In [see text], the nucleotides U(34)U(35)C(36)A(37)C(38) are required for optimal gamma-toxin cleavage, whereas a purine at position 32 or a G in position 33 dramatically reduces the cleavage of the ASL. Comparing modified and partially modified forms of E. coli and yeast [see text] reinforced the strong stimulatory effects of the mcm(5) group, revealed a weak positive effect of the s(2) group and a negative effect of the bacterial 5-methylaminomethyl (mnm(5)) group. The data underscore the high specificity of this yeast tRNA toxin.     

Variant minihelix RNAs reveal sequence-specific recognition of the helical tRNA(Ser) acceptor stem by E.coli seryl-tRNA synthetase.

Aminoacylation rate determinations for a series of variant RNA minihelix substrates revealed that Escherichia coli seryl-tRNA synthetase (SerRS) recognizes the 1--72 through 5--68 base pairs of the E.coli tRNA(Ser) acceptor stem with the major recognition elements clustered between positions 2--71 and 4--69. The rank order of effects of canonical base pair substitutions at each position on kcat/Km was used to assess the involvement of major groove functional groups in recognition. Conclusions based on the biochemical data are largely consistent with the interactions revealed by the refined structure of the homologous Thermus thermophilus tRNA(Ser)-SerRS complex that Cusack and colleagues report in the accompanying paper. Disruption of an end-on hydrophobic interaction between the major groove C5(H) of pyrimidine 69 and an aromatic side chain of SerRS is shown to significantly decrease kcat/Km of a minihelix substrate. This type of interaction provides a means by which proteins can recognize the binary information of 'degenerate' sequences, such as the purine-pyrimidine base pairs of tRNA(Ser). The 3--70 base pair is shown to contribute to recognition by SerRS even though it is not contacted specifically by the protein. The latter effect derives from the organization of the specific contacts that SerRS makes with the neighboring 2--71 and 4--69 acceptor stem base pairs.     

Primary structure of E. coli alanine transfer RNA: relation to the yeast phenylalanyl tRNA synthetase recognition site

Nucleotide sequence comparison of tRNAs aminoacylated by yeast phenylalanyl tRNA synthetase (PRS) have lead to the proposal that the specific nucleotides of the dihydrouridine (diHU) stem region and adenosine at the fourth position from the 3′ end are involved in the PRS recognition site. Kinetic analysis and enzymatic methylation have shown that the size of the diHU loop and the methylation of guanine at position 10 from the 5′ end both directly affect the PRS aminoacylation kinetics. $\text{E. coli}$ tRNA₁^A1a, which is aminoacylated by PRS, should therefore have 1- the specific nucleotides of the diHU stem region and, 2- adenosine at position 4 from the 3′ end. The PRS aminoacylation kinetics of this tRNA indicates that this molecule 3- has a diHU loop of 8 nucleotides and 4- has an unmethylated guanine at position 10 from the 5′ end. We report here the complete sequence of $\text{E. coli}$ tRNA₁^A1a and confirmation of each of these four predictions.     

The T-arm of tRNA is a substrate for tRNA (m5U54)-methyltransferase

Fragments of Escherichia coli FUra-tRNA(1Val) as small as 15 nucleotides form covalent complexes with tRNA (m5U54)-methyltransferase (RUMT). The sequence essential for binding includes position 52 of the T-stem and the T-loop and extends toward the 3' acceptor end of FUra-tRNA. The in vitro synthesized 17mer T-arm of E. coli tRNA(1Val), composed of the seven-base T-loop and 5-base-pair stem, is a good substrate for RUMT. The Km is decreased 5-fold and kcat is decreased 2-fold compared to the entire tRNA. The T-arm structure could be further reduced to an 11mer containing the loop and two base pairs and still retain activity; the Km was similar to that of the 17mer T-arm, whereas kcat was decreased an additional 20-fold. The data indicate that the primary specificity determinants for the RUMT-tRNA interaction are contained within the primary and secondary structure of the T-arm of tRNA.     

Involvement of protein kinase C in the control of tRNA modification with queuine in HeLa cells.

The eukaryotic tRNA:guanine transglycosylase (TGT) catalyses the base-for-base exchange of guanine for queuine (the q-base)--a nutrition factor for eukaryotes--at position 34 of the anticodon of tRNAsGUN (where 'N' represents one of the four canonical tRNA nucleosides), yielding the modified tRNA nucleoside queuosine (Q). This unique tRNA modification process was investigated in HeLa cells grown under either aerobic (21% O2) or hypoxic conditions (7% O2) after addition of chemically synthesized q-base to q-deficient cells. While the q-base was always inserted into tRNA under aerobic conditions, HeLa cells lost this ability under hypoxic conditions, however, only when serum factors became depleted from the culture medium. The inability to insert q into tRNA did not result from a lack of substrate, because the q-base accumulated within these cells against the concentration gradient, suggesting the presence of an active transport system for this base in HeLa cells. The activity of the TGT enzyme was restored after treatment of the cells with the protein kinase C activator, TPA, even in the presence of mRNA or protein synthesis inhibitors. The results indicate that the eukaryotic tRNA modifying enzyme, TGT, is a downstream target of activated protein kinase C.     

Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase to elucidate the role of serine 103 for enzymatic activity.

The tRNA modifying enzyme tRNA-guanine transglycosylase (TGT) is involved in the exchange of guanine in the first position of the anticodon with preQ1 as part of the biosynthesis of the hypermodified base queuine (Q). Mutation of Ser90 to an alanine in Escherichia coli TGT leads to a dramatic reduction of enzymatic activity (Reuter, K. et al. (1994) Biochemistry 33, 7041-7046). To further clarify the role of this residue in the catalytic center, we have mutated the corresponding Ser103 of the crystallizable Zymomonas mobilis TGT into alanine. The crystal structure of a TGT(S103A)/preQ1 complex combined with biochemical data presented in this paper suggest that Ser103 is essential for substrate orientation in the TGT reaction.     

Influence of transfer RNA tertiary structure on aminoacylation efficiency by glutaminyl and cysteinyl-tRNA synthetases

The position of the tertiary Levitt pair between nucleotides 15 and 48 in the transfer RNA core region suggests a key role in stabilizing the joining of the two helical domains, and in maintaining the relative orientations of the D and variable loops. E. coli tRNA(Gln) possesses the canonical Pu15-Py48 trans pairing at this position (G15-C48), while the tRNA(Cys) species from this organism instead features an unusual G15-G48 pair. To explore the structural context dependence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed and evaluated as substrates for glutaminyl and cysteinyl-tRNA synthetases. The glutaminylation efficiencies of these mutant tRNAs are reduced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the tertiary core of this tRNA plays a role in GlnRS recognition. Introduction of tRNA(Cys) identity nucleotides at the acceptor and anticodon ends of tRNA(Gln) produced a tRNA substrate which was efficiently aminoacylated by CysRS, even though the tertiary core region of this species contains the tRNA(Gln) G15-C48 pair. Surprisingly, introduction of G15-G48 into the non-cognate tRNA(Gln) tertiary core then significantly impairs CysRS recognition. By contrast, previous work has shown that CysRS aminoacylates tRNA(Cys) core regions containing G15-G48 with much better efficiency than those with G15-C48. Therefore, tertiary nucleotides surrounding the Levitt pair must significantly modulate the efficiency of aminoacylation by CysRS. To explore the detailed nature of the structural interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bound to GlnRS. These structures show that the larger purine ring of G48 is accommodated by rotation into the syn position, with the N7 nitrogen serving as hydrogen bond acceptor from several groups of G15. The G15-G48 conformations differ significantly compared to that observed in the native tRNA(Cys) structure bound to EF-Tu, further implicating an important role for surrounding nucleotides in maintaining the integrity of the tertiary core and its consequent ability to present crucial recognition determinants to aminoacyl-tRNA synthetases.     

Antideterminants present in minihelix(Sec) hinder its recognition by prokaryotic elongation factor Tu.

During protein biosynthesis, all aminoacylated elongator tRNAs except selenocysteine-inserting tRNA Sec form ternary complexes with activated elongation factor. tRNA Sec is bound by its own translation factor, an elongation factor analogue, e.g. the SELB factor in prokaryotes. An apparent reason for this discrimination could be related to the unusual length of tRNA Sec amino acid-acceptor branch formed by 13 bp. However, it has been recently shown that an aspartylated minihelix of 13 bp derived from yeast tRNA Asp is an efficient substrate for Thermus thermophilus EF-Tu-GTP, suggesting that features other than the length of tRNA Sec prevent its recognition by EF-Tu-GTP. A stepwise mutational analysis of a minihelix derived from tRNA Sec in which sequence elements of tRNA Asp were introduced showed that the sequence of the amino acid- acceptor branch of Escherichia coli tRNA Sec contains a specific structural element that hinders its binding to T.thermophilus EF-Tu-GTP. This antideterminant is located in the 8th, 9th and 10th bp in the acceptor branch of tRNA Sec, corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem. The function of this C7.G66/G49.U65/C50.G64 box was tested by its transplantation into a minihelix derived from tRNA Asp, abolishing its recognition by EF-Tu-GTP. The specific role of this nucleotide combination is further supported by its absence in all known prokaryotic elongator tRNAs.     

Evolution of eukaryal tRNA-guanine transglycosylase: insight gained from the heterocyclic substrate recognition by the wild-type and mutant human and Escherichia coli tRNA-guanine transglycosylases

The enzyme tRNA-guanine transglycosylase (TGT) is involved in the queuosine modification of tRNAs in eukarya and eubacteria and in the archaeosine modification of tRNAs in archaea. However, the different classes of TGTs utilize different heterocyclic substrates (and tRNA in the case of archaea). Based on the X-ray structural analyses, an earlier study [Stengl et al. (2005) Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. Chembiochem, 6, 1926-1939] has made a compelling case for the divergent evolution of the eubacterial and archaeal TGTs. The X-ray structure of the eukaryal class of TGTs is not known. We performed sequence homology and phylogenetic analyses, and carried out enzyme kinetics studies with the wild-type and mutant TGTs from Escherichia coli and human using various heterocyclic substrates that we synthesized. Observations with the Cys145Val (E. coli) and the corresponding Val161Cys (human) TGTs are consistent with the idea that the Cys145 evolved in eubacterial TGTs to recognize preQ(1) but not queuine, whereas the eukaryal equivalent, Val161, evolved for increased recognition of queuine and a concomitantly decreased recognition of preQ(1). Both the phylogenetic and kinetic analyses support the conclusion that all TGTs have divergently evolved to specifically recognize their cognate heterocyclic substrates.