tRNA Recognition of tRNA-guanine Transglycosylase from a Hyperthermophilic Archaeon, Pyrococcus horikoshii*

Masakatsu WatanabeDagger §, Nobukazu NamekiDagger §, Mami Matsuo-TakasakiDagger , Susumu Nishimura||, and Norihiro OkadaDagger **

From the Dagger  Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 and the || Banyu Tsukuba Research Institute (Merck), 3 Ookubo, Tsukuba 300-2611, Japan

Received for publication, June 12, 2000, and in revised form, October 11, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the biosynthesis of archaeosine, archaeal tRNA-guanine transglycosylase (TGT) catalyzes the replacement of guanine at position 15 in the D loop of most tRNAs by a free precursor base. We examined the tRNA recognition of TGT from a hyperthermophilic archaeon, Pyrococcus horikoshii. Mutational studies using variant tRNAVal transcripts revealed that both guanine and its location (position 15) were strictly recognized by TGT without any other sequence-specific requirements. It appeared that neither the global L-shaped structure of a tRNA nor the local conformation of the D loop contributed to recognition by TGT. A minihelix composed of the acceptor stem and D arm of tRNAVal, designed as a potential minimal substrate, failed to serve as a substrate for TGT. Only a minihelix with mismatched nucleotides at the junction between the two domains served as a good substrate, suggesting that mismatched nucleotides in the helix provide the specific information that allows TGT to recognize the guanine in the D loop. Our findings indicate that the tRNA recognition requirements of P. horikoshii TGT are sufficiently limited and specific to allow the enzyme to recognize efficiently any tRNA species whose structure is not fully stabilized in an extremely high temperature environment.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Among the large number of modified nucleosides found in tRNAs, only two hypermodified nucleotides have a 7-deazaguanosine structure, namely, queuosine and archaeosine. Queuosine (Q1; 7-(((4,5-cis-dihydroxy-2-cyclopenten-1-yl)amino)methyl)-7-deazaguanosine) has been found at position 34, the wobble position of the anticodon, of four tRNAs with the anticodon sequence GUN (specific for amino acids Asp, Asn, His, and Tyr), but it has been found in most eubacteria and eukaryotes (1-3). Queuosine is involved not only in the fine tuning of translation in eubacteria, as suggested by its location at the wobble position, but also in cellular events such as development, differentiation, aging, and cancer in eukaryotes. However, the mechanisms responsible for such phenomena are not fully understood (for reviews, see Refs. 4 and 5). Archaeosine (G*; 7-formamidino-7-deazaguanosine) has been found at position 15 in the D loop of tRNAs from many archaea (6, 7). In Haloferax volcanii, the archaeon that has been examined in the greatest detail, archaeosine has been identified in more than 15 tRNA species (8, 9), but its role remains unknown.

A characteristic feature of the biosynthesis of both queuosine and archaeosine is the base replacement reaction that is catalyzed by tRNA-guanine transglycosylase (TGT) (10-13). Eubacterial TGTs catalyze the replacement of guanine at position 34 by the free precursor, 7-aminomethyl-7-deazaguanine (preQ1), and archaeal TGT catalyzes that at position 15 by 7-cyano-7-deazaguanine (preQ0). Subsequent modification is catalyzed by two different enzymes to yield the final products in Escherichia coli (14, 15), whereas such reactions are presumably catalyzed by at least one as yet unidentified enzyme in archaea. By contrast, eukaryotic TGT recognizes the Q base directly and incorporates it at position 34 of those four tRNA species, although it can also recognize both preQ1 and preQ0 in vitro (16, 17). In higher organisms, the Q residues in tRNAAsp and tRNATyr are further glycosylated with mannose and galactose, respectively (18, 19).

The crystal structure of TGT from Zymomonas mobilis in a complex with preQ1 revealed the molecular mechanism of the base replacement reaction as well as the structure of the TGT (20). Furthermore, recent comparisons of the sequences of prokaryotic, eukaryotic, and archaeal TGTs revealed that all TGTs can adopt a structure with a common fold, and that conservation of catalytic residues strongly favors the hypothesis that the mechanism of catalysis by all TGTs is identical (21). Thus, although the mechanism by which a guanine base within a tRNA is replaced by derivatives of 7-deazaguanine seems to have been substantially conserved among kingdoms, there are differences in the sites of incorporation in the substrate tRNA, which probably reflect the specific biological functions of queuosine and archaeosine.

The tRNA recognition properties of eubacterial TGT have been most investigated in vitro (22, 23). A five-base pair minihelix that corresponds to the anticodon arm serves as an efficient substrate for TGT from E. coli, with only a 29-fold decrease in Kcat/Km as compared with the value for a full-length tRNA (22). This result indicates that the L-shaped structure of tRNA is not necessary for recognition by E. coli TGT. Mutational studies using the minihelix revealed that the 33UGU35 sequence in the anticodon loop is a major determinant for tRNA recognition by TGT, and the position 32, adjacent to the UGU sequence, is required to be a pyrimidine. These results are reasonable because all Q-containing tRNAs have G34 and U35 at the first and second positions of the anticodon and, moreover, Y32 and U33 are highly conserved in almost all tRNAs. In eukaryotes, tRNA recognition by TGT was studied in vivo using variants of yeast tRNAAsp and tRNAPhe that were microinjected into Xenopus laevis oocytes (24, 25). The 34UGU36 sequence in the anticodon loop was essential for tRNA recognition by X. laevis TGT, as was the case for E. coli TGT. However, X. laevis TGT had a requirement for the intact architecture of its tRNA substrate that was even more stringent than that of E. coli TGT.

Although eubacterial and eukaryotic TGTs have been well characterized biochemically and/or structurally, little is still known about archaeal TGTs. In this study, we focused on the tRNA recognition by archaeal TGT. There appear to be two types of TGT in terms of the site of action in a tRNA substrate. The reaction site for eubacterial and eukaryotic TGTs is G34 in the anticodon loop, whereas that of archaeal TGT is G15 in the D loop. In the three-dimensional structure of a tRNA, however, the nucleotide at position 15 generally interacts with the nucleotide at position 48 in the variable loop to participate in formation of the central core of the characteristic L-shaped structure (26). In addition, a specific feature of all archaeal tRNAs examined to date is a G15-C48 pair, whereas tRNAs from other organisms have either a G15-C48 or an A15-U48 pair. Furthermore, eubacterial and eukaryotic TGTs recognize only tRNAs specific for Asp, Asn, His, and Tyr, whereas it seems likely that archaeal TGTs recognize most tRNA species, as indicated by the sequences of tRNAs from H. volcanii (8).

In this study, we investigated tRNA recognition by TGT from Pyrococcus horikoshii, using a number of variants of the T7 transcripts of a full-length tRNAs and minihelices. P. horikoshii is a hyperthermophilic archaeon that grows optimally at 98 °C (27). The gene for TGT from this microorganism was cloned and overexpressed in E. coli. Our mutational studies revealed the tRNA recognition requirements of this archaeal TGT, providing an example of the tRNA recognition mode of a hyperthermophilic enzyme whose substrate tRNA cannot form a stable L-shaped structure as a consequence of the high growth temperature. Furthermore, our results, together with those from other organisms, allowed us to make detailed comparisons in tRNA recognition modes that reveal the evolution of tRNA recognition by TGTs.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents, Enzymes, and Plasmids-- Reagents and restriction enzymes were purchased from TAKARA (Japan) or WAKO (Japan), unless otherwise noted. The plasmid pET 3a was obtained from Novagen. [8-14C]Guanine hydrochloride (1.97 GBq/mmol) was obtained from PerkinElmer Life Sciences.

Preparation of Template DNAs and in Vitro Transcription-- A DNA fragment containing the gene for tRNAVal from P. horikoshii, a T7 promoter at the 5'-end, and an MvaI site at the 3'-end was constructed by polymerase chain reaction (PCR) using only two synthetic DNA oligomers with 11-mer complimentary regions. The forward oligomer (5'-ATGTAATACGACTCACTATAGGGCCCGTGGTCTAGTTGGTCATGACGCCGCCCTT-3') and the reverse oligomer (5'-CCACCTGGTGGGCCCGCGGGGATTCGAACCCCGGACCTCCGCCTCGTAAGGGCGGCGT-3') were annealed via their complementary regions (underlined) and amplified by PCR (95 °C for 1 min, 60 °C for 1 min, 72 °C for 30 s; 5 cycles). The products of PCR were ligated into pT7 Blue, and then E. coli strain JM109 was transformed with the resultant plasmid. The sequence of the insert was confirmed by sequencing. Using this plasmid as template, we performed PCR with the following primers, M4 (5'-GTTTTCCCAGTCACGAC-3') and RV (5'-CAGGAAACAGCTATGAC-3'), and then we digested the amplified DNA with MvaI to generate the CCA end of the tRNA. Transcripts of the template DNA were prepared with a T7 runoff-transcription system as described elsewhere (28). Mutant tRNAs and minihelices were prepared similarly.

Amplification of the tgt Gene by PCR and Insertion into an Overexpression Vector-- A DNA fragment containing the tgt gene was amplified, using the primers given below, from the genomic DNA of P. horikoshii strain OT3, which was kindly provided by Dr. I. Matsui of the National Institute of Bioscience and Human Technology, Japan. The forward primer (5'-CCAGCCCATATGAGCCGGGGTGATAAAATGCT-3') contained an NdeI site at its 5'-end, and the reverse primer (5'- GGATCGGATCCGCTTATACCACCAATATTAAAGTCCA-3') contained a BamHI site at its 5'-end. After the reaction mixtures had been incubated at 95 °C for 3 min, amplification by PCR was performed. The amplified DNA was digested with NdeI and BamHI, and the resultant fragment was ligated into pET 3a, an overexpression vector, to yield Phtgt/pET3a.

Overexpression and Purification of TGT-- E. coli BL21(DE3) cells were transformed with the recombinant plasmid Phtgt/pET3a, and the transformants were cultivated at 37 °C in LB medium (1% tryptone, 0.5% NaCl, 0.5% yeast extract) that contained 50 µg/ml ampicillin. After induction with isopropyl-1-thio-beta -D-galactopyranoside (final concentration, 1 mM) at A600 ~ 0.8 for 4 h, the cells were harvested by centrifugation (5000 × g for 20 min, at 4 °C). Approximately 7 g of cells was obtained from 1 liter of culture and stored at -80 °C until use. A cell-free extract was prepared by sonication in 50 volumes (w/w) of buffer A (50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and 0.5 mM EDTA), which contained 400 mM NaCl, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride and 50 µg/µl DNase I (Sigma Chemical Co.). The crude extract was heated at 85 °C for 30 min as described elsewhere (29). After centrifugation of the extract at 10,000 × g at 4 °C for 15 min, the supernatant was concentrated with a Centricon 10 filter (Amicon). The solution of crude enzyme was then loaded onto a Superdex 200 column (16 mm inner diameter × 600 mm; Amersham Pharmacia Biotech, Sweden) and eluted with buffer A that contained 200 mM NaCl. Fractions with enzymatic activity were dialyzed against buffer A, which contained 50 mM NaCl and 1 mM DTT. After dialysis, the sample was applied to a POROS HQ/M column (4.6 mm inner diameter × 100 mm; PerSeptive Biosystems). The enzyme was eluted with a linear gradient of NaCl from 0 to 500 mM in buffer A. Fractions with TGT activity were concentrated with a Centricon 10 filter and examined by SDS-polyacrylamide gel electrophoresis (see Fig. 1). The TGT fraction was stable for at least 6 months when stored at 4 °C.

Assay of TGT Activity in Vitro-- A guanine incorporation assay (TGT assay) was used to determine the kinetic parameters of reactions with tRNA transcripts and minihelices essentially as described elsewhere (30). The reaction was allowed to proceed at 65 °C in a 60-µl reaction mixture that contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 400 mM NaCl, 1 mM DTT, and 15 µM [8-14C]guanine hydrochloride, with various concentrations of transcripts and TGT. Initial rates of base replacement activity were determined at six concentrations of tRNA transcripts, from 0.10 to 5.0 µM, or of minihelices, from 1.5 to 7.0 µM, at a fixed concentration of the enzyme. The TGT concentration for reaction with tRNA transcripts was 11 nM, whereas that for minihelix transcripts is 330 nM. Km and Kcat were determined by plotting of [S] against [S]/v (where [S] is the concentration of tRNA and v is the observed initial velocity of the base replacement reaction; see Fig. 2). We found that charges in the radiolabeled guanine from 15 to 75 µM had no effect on the TGT activities at 65 °C (data not shown).

Melting Profiles of Transcripts-- Absorbance at 260 nm and melting temperatures were monitored in a spectrophotometer (DU-640; Beckman) equipped with a temperature regulator and a six-cell holder. Measurements were made in 50 mM Tris-HCl (pH 7.5) buffer that contained 400 mM NaCl and 5 mM MgCl2. Before the analysis of melting temperature, transcripts were heated to 85 °C for 3 min in the absence of both monovalent and divalent cations and then they were cooled down quickly on ice. Melting profiles are not shown.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

All TGTs characterized to date, irrespective of their sources, can recognize free guanine in vitro and incorporate it into tRNA by a base replacement reaction. This capacity is the basis for a convenient assay of TGT activity, in which the incorporation of radiolabeled guanine into tRNA is monitored (10, 12). To investigate tRNA recognition by TGT from archaea, we investigated the effects on the base replacement reaction of variants of tRNAVal transcripts from P. horikoshii using T7 RNA polymerase. We overexpressed TGT from P. horikoshii in E. coli and purified it by heat treatment at 85 °C for 30 min and subsequent chromatography on columns of Superdex 200 and HQ/M. The resulting fractions contained only a single protein with a molecular mass of 60 kDa, as determined by SDS-polyacrylamide gel electrophoresis (Fig. 1).



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Fig. 1.   Results of purification of TGT from P. horikoshii that had been overexpressed in E. coli. The purified enzyme was subjected to electrophoresis on a denaturing 10% polyacrylamide gel. The left lane (M) was loaded with marker proteins, as indicated.

The wild-type transcript of P. horikoshii tRNAVal was an excellent substrate for TGT, with a Km of 0.57 µM and a Kcat of 8.2 × 10-2 s-1 (Fig. 2A, Table I). The low turnover rate of TGT of <1/min was in accordance with the multistep mechanism of the base replacement reaction, as proposed for bacterial TGT (20). A low Kcat was also reported for E. coli TGT (22, 30).



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Fig. 2.   Kinetics of base replacement activities by TGT for (A) full-length wild-type tRNAVal transcript and (B) a minihelix (with insertion of A between 25 and 26; see Fig. 5).


                              
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Table I
Kinetic parameters of reactions catalyzed by TGT with full-length wild-type and variant tRNA transcripts

TGT Is Most Active at Extremely High Temperatures-- P. horikoshii grows at temperatures between 80 and 102 °C, with optimum growth at 98 °C (27). The few enzymes from this organism that have been characterized to date are highly thermophilic and active at high temperatures (29, 31). Thus, most of other enzymes from P. horikoshii might be expected to have similar properties. To examine the effects of temperature on TGT activity, we varied the reaction temperature from 35 to 100 °C (Fig. 3). Our results showed that the enzyme was most active at 100 °C. To verify the thermal stability of the substrate, we determined the melting temperature of the transcript of P. horikoshii tRNAVal. It was lower (79 °C) than the optimum temperature for the reaction catalyzed by TGT. Accordingly, in this study, we fixed the temperature for the assays at 65 °C, taking into consideration both the efficiency of the enzymatic reaction and the thermal stability of tRNA transcripts.



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Fig. 3.   Effects of temperature on TGT activity. The reaction was allowed to proceed at each temperature for 4 min in a 60-µl reaction mixture that contained 3 µM wild-type tRNAVal transcript and 65 nM TGT. Other details are given under "Experimental Procedures."

The Correct L-shaped Structure of tRNA Is Not Required for tRNA Recognition by TGT-- Several invariant nucleotides that are crucial for the canonical L-shaped structure of a tRNA are located within the D and T loops (26) (Fig. 4). G18 and G19 in the D loop interact with U55 and C56 in the T loop, respectively, and these interactions are responsible for the formation of the L-shaped structure. These base pairings are important for tRNA recognition by some tRNA-modifying enzymes (25). Substitution of 18GG19 by CC or of 55UC56 by GG in the tRNAVal transcripts yields a slightly better substrate than the wild-type tRNAVal transcript (Tables I and II). The U8-A14 interaction is also crucial for the proper folding of tRNAs. Substitution of U8 by A or of A14 by C had no effect on the efficiency of the reaction. These results indicated that neither the tertiary base pair interactions nor the correct L-shaped structure is involved in tRNA recognition by TGT.



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Fig. 4.   A, schematic representation of the cloverleaf structure of tRNAVal from P. horikoshii. Numbering is based on that of a previous study (44). Arrows indicate the substitutions made in this study. The substitutions indicated by italic letters, of which the data are not shown, had no effects on the efficiencies of TGT activity. B, conserved and semiconserved nucleotides in tRNAs from P. horikoshii. Uppercase and lowercase letters indicate conserved and semiconserved nucleotides, respectively. R, Y, and S represent purine, pyrimidine, and either G or C, respectively. The black bold dots indicate nonconserved nucleotide. The thin lines represent tertiary base pairs. The alpha  and beta  regions are defined in the text.

No Specific Bases in the D Loop Are Required for Recognition with the Exception of G15-- In the tRNAs of P. horikoshii, G15 in the D loop and C48 in the variable loop are both conserved nucleosides (32). Irrespective of the modification of G15 or lack of it, G15 can make a tertiary Levitt base pair with C48 to participate in formation of the central core of the tRNA structure. Substitution of G15 by any other nucleotide resulted in the complete absence of detectable base replacement activity, whereas that of C48 by any other nucleotide resulted in higher activity than with the wild-type substrate, with an increase in Kcat (Tables I and II). As for A59 in the T loop that stacks with the G15-C48 pair, the efficiency of the reaction was unaffected by substitution of A59 by any other nucleotide. The mutant with deletion of U47 in the variable loop was also a good substrate, even though the bulged nucleotide at position 47 often plays an important steric role, allowing the canonical tertiary interactions 15-48 and 22-46 (33). Our results showed that guanosine at position 15 was strictly recognized by TGT, whereas neither the Levitt base pair G15-C48 nor C48 itself contributed to recognition by TGT.

The D loop has variable regions between G15 and 18GG19 (the alpha  region) and between 18GG19 and A21 (the beta  region), respectively (34). The number of nucleotides in the alpha  and beta  regions, as well as the nature of the nucleotides, varies depending on the tRNA species (Fig. 4). We generated mutants with all combinations of nucleotide substitutions in the alpha  region to examine whether the efficiency of the base replacement reaction might depend on a particular nucleotide combination in the alpha  region. No significant effects were observed with any of the combinations tested (Table II). In the beta  region, deletion of the two nucleotides U20 and C20a did not affect activity. These results, together with the results of mutation of A14 and 18GG19, showed that no sequences in the D loop were recognized by TGT with the exception of G15 and, simultaneously, that the length of the D loop, the alpha  region, or the beta  region was not involved in the tRNA recognition.


                              
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Table II
Relative activities of TGT with full-length wild-type and variant tRNA transcripts
Each reaction was allowed to proceed for 12 min at 65 °C in a 60-µl reaction mixture that contained 1 µM wild-type or mutant tRNAVal transcript and 65 nM P. hurikoshii TGT unless otherwise noted under "Experimental Procedures."

The D Stem Is Not Involved in tRNA Recognition-- The D stem has either 3 or 4 base pairs. Substitution of U13 by A, which probably increased the number of base pairs from 3 to 4, had no effect on the efficiency of the reaction. Substitution of C12 by G, which probably shortened the stem, resulted in higher activity than that with the wild-type substrate (Tables I and II). Although these disruptions of the D stem would be expected to perturb the L-shaped structure considerably, the deformed structures appeared to favor recognition by TGT, as in the case of mutations that led to disruption of tertiary base pair. Additional disruption of base pairs, namely, substitution of 11UC12 by 11AG12, decreased the activity by only 50%. Compensatory base pair changes, namely, from G10-C25 to C-G, from U11-A24 to A-U, and from C12-G23 to G-C, had no effects, even though they probably affected tertiary interactions of the D stem with position 9 or with the variable loop (Fig. 4B). These results indicated that neither the D stem itself nor the number of base pairs was essential for the recognition of its tRNA substrate by TGT.

A Guanosine Residue and Its Location in the D Loop Are Essential for the tRNA Recognition-- The location of the guanosine residue in the D loop was altered from position 15 to positions 14 or 16. A14 and G15 were changed to G14 and U15, respectively, to move guanine from position 15 to 14. This mutation led to a major decrease in the TGT activity, with a 3700-fold decrease in Kcat/Km (Tables I and II). Similarly, the mutant in which G15 and U16 were changed to A15 and G16 resulted in a 2200-fold decrease in Kcat/Km. The possibility that these large decreases were due to disruption of the tertiary base pairs U8-A14 and G15-C48 could be excluded in view of the apparent unimportance in recognition of the L-shaped structure of tRNA. These findings indicated that both the guanosine residue and its location (position 15 from the 5'-end) in tRNA were strictly recognized by TGT and suggested the presence of a specific reference site in the tRNA structure by which the guanosine residue and its location were recognized. It is noteworthy, in this context, that there is no guanosine residues in the vicinity of position 15 in the D loop of P. horikoshii tRNAs: position 14 is occupied by an invariant nucleotide, A14, and position 16 is often a pyrimidine or, in a few tRNAs, adenine (32).

Neither the Anticodon Stem Nor the T Stem Contribute to tRNA Recognition-- We introduced mutations into the region of the anticodon and T stems (Fig. 4A). A change from 27CCGCC31 to 27GGCGG31 was made in the anticodon stem such that the original stem was seriously disrupted. A similar change from 61CCCCG65 to 61GGGGC65 was made in the T stem. Although these large substitutions would appear to have significant effects on the entire tRNA structure, the former change resulted in only a 1.4-fold decrease in Kcat/Km, whereas the latter resulted in a 4.3-fold decrease in Kcat/Km, with the major effects in both cases being on Km (Tables I and II). These results showed that neither the anticodon stem nor the T stem played any substantial role in tRNA recognition, leading to the suggestion that a simpler and smaller structure might be capable of serving as a substrate for TGT.

A Normal Minihelix Cannot Be a Substrate for TGT, but a Minihelix with Mismatched Bases Can Serve as a Substrate-- To elucidate the requirements for recognition of G15 in the D loop by TGT, we constructed a minihelix composed of the D arm and the acceptor stem as a model substrate (Fig. 5). This normal minihelix had no detectable substrate activity, and its melting temperature was 87 °C. However, when adenine was inserted at the junction between the D stem and the acceptor stem (positions 25 and 26), the new minihelix mutant served as a substrate for TGT with only a 240-fold decrease in Kcat/Km compared to that of the wild-type tRNA substrate (Fig. 2B, Table III). Similar results were obtained with a minihelix in which guanine was inserted instead of adenine. We also inserted adenine between positions 6 and 7, 8 and 9, 9 and 10, and 26 and 27, respectively, and we detected TGT activity only with the minihelix in which adenine had been inserted between positions 26 and 27. In this case, there was a 340-fold decrease in Kcat/Km relative to that of the wild-type tRNA substrate. These findings indicated the importance of the site of insertion, but not of the nature of the inserted nucleotide, for conversion of the original minihelix to be an effective substrate for TGT.



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Fig. 5.   Schematic representation of the minihelix composed of the D arm and the acceptor stem of P. horikoshii tRNAVal. Arrows indicate the positions of insertion of nucleotides in this study. Boxed letters indicate nucleotides that were substituted by other nucleotides.


                              
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Table III
Kinetic parameters of reactions catalyzed by TGT with minihelices as substrates

Instead of inserting an additional nucleotide, we introduced a non-Watson-Crick base pair into the stem of the minihelix. Replacement of G10 by A, which created an A10-C25 pair in the helix, stimulated base replacement activity, with a 160-fold decrease in Kcat/Km. A minihelix with A9-C26, in which G9 had been replaced by A9, yielded a 270-fold decrease in Kcat/Km. In contrast, the minihelices with C9-A26, G10-U25, and C10-A25 combinations were even worse substrates, yielding 2200-, 2600-, and 530-fold decreases in Kcat/Km, respectively. Starting with the minihelix with the A10-C25 pair, we introduced several mutations into the D loop of the minihelix. Substitution of G15 by U15 resulted in the absence of detectable TGT activity, which indicated that G15 in the helix was the site of incorporation by P. horikoshii TGT. The location of guanine in the D loop was changed to position 14 or 16 in the D loop. Substitution of A14 and G15 by G14 and U15, or of G15 and U16 by A15 and G16 also resulted in the absence of detectable activity. These results were essentially the same as those obtained with full-length tRNA transcripts.

We also made several minihelices in which we increased the number of adenine residues between positions 25 and 26. Because the U9-A26 base pair is flanked by more than two G-C base pairs, insertions at position 26 seemed unlikely to generate an unexpected structure. Insertion of three adenine residues at this site resulted in stimulation of activity, with only a 49-fold decrease in Kcat/Km as compared with that obtained with the wild-type tRNA. Insertion of five adenine residues yielded the highest base replacement activity of all minihelices tested, with only a 22-fold decrease in Kcat/Km.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recognition of tRNA by TGT-- This study revealed that both a guanosine residue and its location (position 15) in the tRNAVal transcript were strictly recognized by TGT from P. horikoshii without any other sequence-specific requirements. Neither a global L-shaped structure of tRNA nor the local conformation of the D loop within the tRNA appeared to be involved in recognition of its substrate by TGT. The guanosine residue at position 15 is, indeed, located in the D loop in the secondary structure, but it also makes a Levitt base pair with C48 in three dimensions. Thus, it is likely that, before the base replacement reaction can occur, this base pair must be disrupted to expose the guanine base as required for recognition by the enzyme. This hypothesis is supported by the results with mutants with the disrupted tertiary base pairs, which were better substrates for TGT than the wild-type. The hypothesis is also supported by the ability of the minihelices to serve as substrates for TGT. It follows that the catalytic process requires a structural change in the tRNA, which involves interaction between the enzyme and position 15. Similar requirements for recognition of the D loop were reported in the case of tRNA (guanosine-2')-methyltransferase from Thermus thermophilus, which recognizes the invariant nucleotide G18. This nucleoside interacts with position 55 to generate the interaction between the D loop and the T loop in the L-shaped folding of tRNA (35).

Recognition of tRNA in which the L-shaped architecture plays no essential role has obvious advantages in view of the extremely high temperature conditions under which P. horikoshii grows optimally. Although P. horikoshii TGT is highly thermophilic and active above 100 °C, tRNAs lacking all modifications are so unstable that they cannot form a correct stable L-shaped structure immediately after transcription at such high temperatures. The melting temperature of the tRNAs without any modifications ranges from 80 to 85 °C, as predicted from the G-C pair contents in tRNAs from P. horikoshii that attain the maximum (~80%) (Ref. 36 and Fig. 4A). According to detailed studies of the molecular mechanism of thermal unfolding of tRNAs, tRNAs unfold in a few discrete steps as the temperature is raised (37, 38). First, the D stem unfolds at the same time as the tertiary structure is lost, followed in succession by the unfolding of the T stem, the anticodon stem and, finally, the acceptor stem. Hence, at high temperatures, tertiary folding and the D stem are the most unstable and thus, as anticipated, neither tertiary folding nor the D stem contributed to recognition of its substrate by P. horikoshii TGT. The recognition mode of this tRNA-modifying enzyme seems likely to have evolved for optimal recognition of a structurally unstable tRNA substrate. Our results also indicate that TGT is one of the enzymes in the tRNA modification pathway that can modify tRNA at an early stage, when a tRNA not only is an untrimmed and unspliced primary transcript but also has not yet folded to yield a correct three-dimensional structure (25).

Although the role of archaeosine is unknown, the position of the modification in the tRNA core suggests a role in the thermal stability of tRNA structure. The absence of base-specific requirements for tRNA recognition, with the exception of G15, probably allows all tRNAs in P. horikoshii to serve as substrates for TGT, unless they have some, as yet unknown, negative recognition element. Consequently, they might all have archaeosine at position 15. In P. furiosus, the melting temperature of unfractionated tRNAs (97 °C) is ~20 °C higher than that predicted solely from the G-C content, and it has been attributed primarily to post-transcriptional modification (39). We propose that the presence of archaeosine in archaeal tRNAs would play a role in promoting and/or stabilizing the folding of precursor tRNAs into a correct tertiary structure, which can then be recognized by those enzymes that recognize their tRNA substrates in ways that depend on the three-dimensional architecture of the tRNAs (25). We should note here that archaeosine is the only modified nucleoside in the D loop of the various archaeal tRNAs that have been examined to date (40).

Recognition of a Minihelix by TGT-- The minihelix with a 12-bp stem composed of the D arm and the acceptor stem was incapable of serving as a substrate for TGT. For recognition by TGT, the minihelix required mismatched nucleotides located exclusively at the junction between the D stem and the acceptor stem. In the case of an A-C pair in a 16-mer RNA duplex, x-ray analysis of the crystal structure at neutral pH and room temperature demonstrated that RNA with an isolated A-C base pair bends by 23° at the wobble site (41). The minihelix with an A-C pair at positions 10-25 or 9-26 was an even better substrate than the minihelix with a C-A pair at either site. These observations suggest that TGT preferentially recognizes an RNA helix that bends in direction, which might be reminiscent of the deformed-helix structure of the acceptor stem and the D arm, linked by two nucleotides, 8UA9, within the three-dimensional structure of tRNA.

We also found that the minihelix that was an effective substrate for TGT required insertion of nucleotides at the junction between the two domains on one strand of the stem exclusively (on the side closer to the 3'-end) and not on the other strand. These nucleotides probably form a bulge. This phenomenon may be explained by one or both of the following scenarios. (i) Nucleotides inserted in the strand closer to the 3'-end, but not the 5'-end, might cause the RNA helix to bend in a specific direction that favors recognition by TGT, as in the case of minihelices with an A-C pair. (ii) Although the location of the guanine residue in the loop might not be altered, insertion into the strand closer to the 5'-end side might lead to a shift in the position of G15 to position 16 relative to the 5'-end, such that the minihelix is no longer recognized by TGT. Further nucleotide insertion experiments showed that the minihelix became a better substrate as the number of inserted nucleotides increased. It is possible that the minihelix with more inserted nucleotides might mimic the deformed helix that consists of the acceptor stem and the D arm within three-dimensional structure of the entire tRNA. Our findings with minihelices suggest that mismatched nucleotides in the helix provide a recognition feature similar to some aspect of the tertiary folding of tRNA, and/or a specific reference site through which TGT locates the guanosine residue in a loop within the secondary structure of tRNA. Recent studies, with resolution at the atomic level, of complexes of proteins with specific RNAs have shown that mismatched nucleotides and bulges confer a significant plasticity on RNA helices and generate grooves wide enough for protein secondary structure to penetrate (for review, see Ref. 42). In several studies of protein-RNA interactions, a bulge of a nucleotide in a helix was shown to serve just as a protein-binding site. The recognition of minihelices by TGT might require the distortion of the regularity of the helices, which can be induced by a mismatched base pair or a bulge.

Similar results with respect to recognition of minihelices have been reported only in the case of human RNase P, which correctly cleaves the extra 5'-region from precursors to tRNAs (43). A minihelix composed of the acceptor stem and the T arm cannot serve as a substrate for human RNase P. However, a minihelix in which nucleotides are inserted into the strand closer to the 5'-end at the junction between the two domains serves as an excellent substrate. These results, together with ours, provide an image of a substrate tRNAs that differs from a cloverleaf with four helices and an L-shaped structure. TGT appears to recognize as its substrate a tRNA that is made up of one helix with a large bulge region that consists of the anticodon arm, the variable loop, and the T stem, whereas human RNase P recognize a tRNA that is made up of one helix with a large bulge region that consists of the anticodon arm, the variable loop, and the D arm (43).

Recognition of tRNAs by TGT from E. coli and P. horikoshii-- Our data allows us to make detailed comparisons with data obtained with E. coli TGT. With respect to base-specific recognition, the 32YUGU35 sequence in the anticodon loop is required by E. coli TGT, whereas only G15 in the D loop is required by P. horikoshii TGT. With respect to recognition of a local conformation, the anticodon-loop structure with the above mentioned sequence is crucial in E. coli, whereas a native D loop structure within an L-shaped tRNA is not necessary in P. horikoshii. With respect to recognition of structure, an L-shaped structure is unnecessary for recognition by either TGT and, thus, minihelices lacking several domains can serve as efficient substrates for both TGTs. However, only the TGT from P. horikoshii requires mismatched bases in the stem of the substrate minihelix.

These comparisons show that requirements for tRNA recognition by E. coli TGT are true of the case in which those by P. horikoshii TGT are made even more strict. It appears that some aspects of tRNA recognition have been conserved between the two types of TGT, albeit with some obvious differences. During evolution, the tRNA recognition mode of TGT seems likely to have changed without much change in the enzyme, with the site of action shifting from position 15 to 34 or vice versa. Divergence of modes of tRNA recognition by tRNA-modifying enzymes would have created opportunities for the creation of new functions for modified nucleotides.


    ACKNOWLEDGEMENTS

We are grateful to Dr. I. Matsui of the National Institute of Bioscience and Human Technology, Japan, for providing genomic DNA of P. horikoshii, and to Prof. T. Ueda of the University of Tokyo for helpful suggestions. We also thank T. Kijimoto, T. Suzuki, S. Tomari, and M. Kajikawa for their technical assistance.


    FOOTNOTES

* This work was supported by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science and Culture, Japan (to N. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These two authors contributed equally to this work.

Present address: Dept. of Industrial Chemistry, Chiba Institute of Technology, Chiba 275-0016, Japan.

** To whom correspondence should be addressed: Tel.: 81-45-924-5742; Fax: 81-45-924-5835; E-mail: nokada@bio.titech.ac.jp.

Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M005043200


    ABBREVIATIONS

The abbreviations used are: Q, queuosine; TGT, tRNA-guanine transglycosylase; preQ0, 7-cyano-7-deazaguanine; preQ1, 7-aminomethyl-7-deazaguanine; PCR, polymerase chain reaction; DTT, dithiothreitol; bp, base pair(s).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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