©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Highly Specific and Efficient Cleavage of Squid tRNA Catalyzed by Magnesium Ions(*)

Mami Matsuo (1), Takashi Yokogawa (1), Kazuya Nishikawa (1), Kimitsuna Watanabe (2), Norihiro Okada (1)(§)

From the (1) Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226, Japan and the (2) Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two lysine isoacceptor tRNAs corresponding to the codons AAA and AAG, respectively, were isolated from squid ( Loligo bleekeri), and their nucleotide sequences were determined. During this analysis, we discovered that the tRNA with the anticodon CUU was efficiently cleaved at a specific site in the presence of magnesium ions, whereas the tRNA with the anticodon UUU was not. Cleavage occurred almost exclusively at the phosphodiester linkage between Gand D(p16). The most remarkable feature of this cleavage reaction is that the end product was not a 2`,3`-cyclic phosphate but was mainly a 3`-phosphate. Thus, this reaction was distinct from the well characterized cleavage of yeast tRNAby lead and from reactions catalyzed by various other metalloribozymes. The presence of a cytidine residue at position 60 was required for efficient cleavage but was not crucial for the reaction, and the entire tRNA molecule had to be intact for this specific and efficient cleavage reaction. The present study provides evidence that there exists a new catalytic mechanism for cleavage of tRNA that exploits biologically ubiquitous ions rather than toxic, nonessential ions such as lead.


INTRODUCTION

The reaction of yeast tRNAwith Pbions at pH 7.0 was the first well characterized example of a metal-promoted site-specific cleavage reaction involving RNA (1, 2, 3) . Cleavage occurs almost exclusively at the phosphodiester linkage between Dand G(p18) (1, 2, 3, 4) , resulting in the formation of a 2`,3`-cyclic phosphate at D(2, 3) . The reaction involves the initial abstraction of a proton from a sugar 2`-OH group at the cleavage site by the ionized lead hydrate, which acts at the metal-binding pocket formed by the D and T loops (2, 3) . Other cations, such as Znand Eu, are also capable of cleaving purified tRNAs at precise locations to yield 2`,3`-cyclic phosphates and 5`-hydroxyl termini (5, 6, 7) .

Although this seemingly anomalous reaction is not known to have biological relevance, the discovery of catalytic RNAs or ribozymes placed this reaction in a broader mechanistic context (8, 9) . In the first step of the self-splicing reaction of the Tetrahymena ribozyme, a Mgor Mnion contributes directly to the reaction by coordination with the 3` oxygen atom in the transition state, presumably stabilizing the developing negative charge on the leaving group (10) . Other ribozymes, such as hammerheads and hairpins, have also been proposed to be essentially metalloribozymes (11, 12) .

On the bases of the above described findings, yeast tRNA, which undergoes intramolecular cleavage in the presence of Pbions, has been redesigned to perform the intermolecular cleavage of RNA as a true enzyme (13, 14) . Furthermore, Pan and Uhlenbeck (15, 16) have used an in vitro selection method to produce a series of lead-dependent ribozymes that efficiently cleave RNA substrates with multiple turnover. One particular variant of the ``leadzyme'' promotes the formation of 2`,3`-cyclic phosphate termini and goes further, catalyzing hydrolysis of the cyclic phosphate to a 3`-terminal phosphate (16) . This two-step mechanism is commonly exploited by several protein ribonucleases, but it is unique among all ribozymes described to date. The many examples of metal-catalyzed cleavages of tRNA described to date are presumed to resemble, in terms of mechanism, the reactions catalyzed by various other metalloribozymes.

In the present report, we provide another example of cation-mediated cleavage of tRNA, in which a Mgion is involved in hydrolysis of a cyclic phosphate to yield a 3`-terminal phosphate, as in the reaction of the ``leadzyme.'' In the present case, an intact tRNA structure must be preserved if the cleavage reaction is to proceed.


EXPERIMENTAL PROCEDURES

Materials

RNase T1, RNase PhyM, and T4 RNA ligase were purchased from Pharmacia Biotech Inc. (Tokyo, Japan); nuclease P1 was from Yamasa Shoyu (Chiba, Japan); RNase U2, guanosine 2`,3`-cyclic monophosphate 5`-monophosphate (designated pG>p), and guanosine 2`,3`-cyclic monophosphate (designated G>p) were from Sigma; and RNase CL3 was from Boehringer Mannheim Yamanouchi (Tokyo, Japan). Alkaline phosphatase from Escherichia coli and T7 RNA polymerase were obtained from Takara Shuzo (Kyoto, Japan). [5`-P]pCp, [-P]UTP, [-P]GTP, and [-P]ATP were from Amersham (Tokyo, Japan). T4 polynucleotide kinase and other chemical reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Purification and Determination of the Nucleotide Sequence of Squid tRNAs

700 g of squid ( Loligo bleekeri) were homogenized in liquid nitrogen. After 3 liters of buffer A (44% (v/v) phenol, 0.5 M NaCl, 0.25 mM EDTA, 50 mM NaOAc, pH 6.0, 10 mM Mg(OAc), 1.2% diethylpyrocarbonate) had been added, the mixture was shaken for 12 h. Total nucleic acids recovered by ethanol precipitation were subjected to chromatography on a column of DE52-cellulose that had previously been equilibrated with buffer B (0.2 M NaCl, 10 mM Mg(OAc), 20 mM NaOAc, pH 6.0).

Bulk tRNA was obtained by stepwise elution with buffer B that contained 0.6 M NaCl in place of 0.2 M NaCl. The unfractionated tRNA (2,000 Aunits) was applied to a column (1.5 45 cm) of DEAE-Sepharose that had previously been equilibrated with buffer C (0.1 M NaCl, 20 mM Tris-HCl, pH 7.6, 8 mM MgCl). The column was washed with buffer C and then eluted with a gradient obtained by placing 1 liter of buffer C in the mixing chamber and 1 liter of buffer C that contained 0.4 M NaCl in place of 0.1 M NaCl in the reservoir. A total of 150 Aunits of the tRNA fraction that contained tRNAwith the anticodon UUU (designated tRNA(UUU)) was obtained. Then, 10 Aunits/experiment were further applied to a column (0.5 10 cm) of RPC-5 that had previously been equilibrated with buffer D (0.4 M NaCl, 10 mM Tris-HCl, pH 7.5, 10 mM Mg(OAc)). The column was washed with buffer D and then eluted with a gradient obtained by placing 20 ml of buffer D in the mixing chamber and 20 ml of buffer D that contained 1 M NaCl in place of 0.4 M NaCl in the reservoir. From an initial 10 Aunits of tRNA applied to the RPC-5 column, a 0.2 Aunit of purified tRNA(UUU) was finally obtained after electrophoresis in a non-denaturating gel.

The tRNA fraction containing the tRNAwith the anticodon CUU (designated tRNA(CUU); 1,200 Aunits) eluted from the column of DEAE-Sepharose was further applied to another column (1 50 cm) of DEAE-Sepharose that had previously been equilibrated with buffer E (0.4 M NaCl, 10 mM MgCl, 20 mM NaOAc, pH 4.0). The column was washed with buffer E and then eluted with a gradient obtained by placing 500 ml of buffer E in the mixing chamber and 500 ml of buffer E that contained 0.75 M NaCl in place of 0.4 M NaCl in the reservoir. A total of 91 Aunits of a tRNA fraction that contained tRNA(CUU) was obtained from the column of DEAE-Sepharose (pH 4.0). Next, 10 Aunits of this fraction/experiment were subjected to chromatography on the same column of RPC-5 as that used for purification of tRNA(UUU), and the column was eluted with a gradient of NaCl under the same conditions as described above. From the 10 Aunits of the tRNA fraction applied to the RPC-5 column, 0.09 Aunit of purified tRNA(CUU) was finally obtained after electrophoresis in a non-denaturing gel.

The fractions containing tRNAsthat were eluted from columns were assayed by aminoacylation using an extract S-100 of hen oviduct and hybridization with oligodeoxyribonucleotide probes specific to the squid genes for tRNA(UUU) and tRNA(CUU) by the previously published methods (17, 18) . The sequences of the two species of tRNAswere determined by the postlabeling method of Kuchino et al. (19) and a partial enzymatic digestion method (20) . Construction of Plasmids That Contained Genes for tRNA(CUU)(C) and tRNA(CUU)(U) and Transcription in Vitro of the DNAs That Included These Genes-Three DNA fragments, namely Ribo-5`, which contains the promoter sequence of T7 RNA polymerase (5`-TAATACGACTCACTATAGCCCGGCTAGCT-3`), Ribo-3`C (5`-CCTGGCGCCCAACGTGGGGCTCGA-3`), and Ribo-3`U (5`-CCTGGCGCCCAACGTGGGACTCGA-3`) were synthesized and used as primers for polymerase chain reaction (21) . Plasmid DNA that included the gene for tRNA(CUU) isolated from a genomic library of the squid() was used as a template for polymerase chain reaction. Using two sets of primers, we amplified and cloned DNAs in PUC19, obtaining pC60K and pU60K, respectively. The pC60K plasmid contained a gene for tRNA(CUU) that had a cytidine residue at position 60 (designated tRNA(CUU)(C)). It generated the same sequence as that of the native tRNA(CUU) of squid except for Uand Aby in vitro transcription. This U-A pair was changed to a G-C pair to allow the plasmid to be transcribed more efficiently. The pU60K plasmid contained the same sequence as that of pC60K except for one nucleotide at position 60; the transcript had a uridine residue at this position (designated tRNA(CUU)(U)).

DNAs were amplified by polymerase chain reaction, using M4 (5`-GTTTTCCCAGTCACGAC-3`) and Reverse (5`-CAGGAAACAGCTATGAC-3`) as primers and pC60K DNA and pU60K DNA, respectively, as templates. The resultant DNA was digested with MvaI to generate the CCA end of tRNA and was transcribed in vitro in a reaction mixture of 100 µl that contained 2 mM of each NTP, 5 mM dithiothreitol, 10 mM MgCl, 1 mM spermidine, 40 mM Tris-HCl, pH 8.1, 50 µg/ml bovine serum albumin, and 100 units of T7 RNA polymerase (22) .

Analyses of Terminal Structures Produced by Cleavages Catalyzed by Metal Ions

For analysis of terminal structures produced by cleavage with Mgions, the 15-mer oligonucleotide was recovered from a denaturing polyacrylamide gel. The 15-mer was digested with RNase U2 in a reaction mixture that contained 0.5 Aunit of guanosine 2`,3`-cyclic phosphate (designated G>p), 10 mM sodium citrate, pH 4.5, and 50 units of RNase U2 in a final volume of 5 µl at 37 °C for 1 h. G>p was included as an internal control to demonstrate that RNase U2 did not cleave a 2`,3`-cyclic phosphate ring under the conditions of the reaction. The digest was subjected to two-dimensional thin-layer chromatography with solvent system A (isobutyric acid, NH, and HO, 66:1:33, v/v) in the first dimension and solvent system B (60 g of ammonium sulfate dissolved in 100 ml of 0.1 M sodium phosphate buffer (pH 6.8) plus 2 ml of n-propyl alcohol) in the second dimension (19) . In the case of digestion with nuclease P1, the 15-mer was incubated in a reaction mixture that contained 1 µg of nuclease P1 and 0.25 Aunit of carrier tRNA in a final volume of 10 µl at 37 °C for 3 h.

For analysis of the terminal structure produced by cleavage with Pbions, the 17-mer oligonucleotide was recovered from a denaturing polyacrylamide gel. The 17-mer was digested with nuclease P1 under the same conditions as used in the case of the 15-mer. In another experiment, for the assay of the phosphomonoesterase activity associated with nuclease P1, the 17-mer was preincubated in a reaction mixture that contained 0.2 N HCl at 37 °C for 1 h in a final volume of 5 µl to open the 2`,3`-cyclic phosphate ring. After neutralization of the mixture by the addition of 0.2 N NaOH, the 17-mer was recovered by ethanol precipitation and was digested with nuclease P1. Solvent systems used for thin-layer chromatography were the same as those used in the case of the analysis of the 15-mer.


RESULTS

Optimization of the Cleavage Reaction

The species of tRNAshaving anticodons CUU and UUU (designated tRNA(CUU) and tRNA(UUU), respectively) were purified to homogeneity. The sequence was first analyzed by a partial enzymatic digestion method (20) and was later confirmed by the postlabeling method of Kuchino et al. (19) for identification of modified nucleotides in the tRNAs. The sequences of the two tRNAs are shown in Fig. 1, in which differences between the two tRNAs are shaded.


Figure 1: Sequences of two species of tRNAs from squid with the anticodons CUU ( A) and UUU ( B), respectively. The nucleotides that differ between these two tRNAs are shaded. N, U*, and A* are unidentified modified nucleotides. N in tRNA(CUU) is probably a derivative of C. The nucleotide at position 54 was shown not to be Um (2`- O-methyluridine) but rather to be Tm (2`- O-methyl ribothymidine). The nucleotide at position 55 is probably a pseudouridine but was not unambiguously identified in the present study. The site of cleavage in the presence of Mgions is indicated by an arrow.



Fig. 2 shows autoradiograms of sequencing polyacrylamide gels for the two tRNAs after partial digestion with base-specific RNases. As shown in Fig. 2( A and B, lanes 1), tRNA(CUU) was cleaved at a specific site even in the absence of any enzyme, whereas tRNA(UUU) was not cleaved under the same conditions. This cleavage reaction was presumed to be due to incubation of the tRNA with a buffer that contained 10 mM Mgions for a certain period of time before sequencing.


Figure 2: Autoradiograms of sequencing polyacrylamide gels for analysis of tRNA(CUU) ( A) and tRNA(UUU) ( B) from squid. After treatment with bacterial alkaline phosphatase of the two tRNAs(about 0.02 Aunit of each), each was labeled at its 5`-end by [-P]ATP and polynucleotide kinase. An aliquot of the labeled tRNA was incubated in the absence ( lane 1) or in the presence of NaCObuffer (pH 9) ( lanes 2 and 7), RNase T1 ( lane 3), RNase U2 ( lane 4), RNase Phy M ( lane 5), or RNase CL3 ( lane 6) according to the protocol described elsewhere (20). The cleavage product of tRNA(CUU) in lane 1 is indicated by a black arrow, and the corresponding position in tRNA(UUU) is indicated by a white arrow. Electrophoresis was performed in 15% polyacrylamide gels that contained 7 M urea and 10% glycerol for 3 h.



We examined the kinetics and the optimum conditions for the cleavage reaction. Fig. 3 A shows the time course of the reaction in the presence of 30 mM MgCl(pH 7.5). In this experiment, native squid tRNA(CUU) labeled with [5`-P]pCp at the 3` terminus was used as a substrate to determine whether there might be another cleavage site in the 3` portion of the tRNA. After incubation for 12 h, about 75% of the tRNA had been cleaved. As shown in lanes 1 and 2 of Fig. 3 B, Mgions were required for the cleavage reaction, and spermidine could not take the place of Mgions. In the absence of Mgions and spermidine, slight cleavages were observed due to degradation of the tRNA (Fig. 3 B, lane 1). However, when spermidine was present, no such degradational cleavages occurred (Fig. 3 B, lane 2). Spermidine may possibly contribute to the maintenance of a stable tertiary structure of the tRNA. The optimal concentration of Mgions was found to be 30 mM, and almost the same extent of cleavage was observed at concentrations of 30 and 50 mM (Fig. 3 B, lanes 7-10). In the presence of Mgions, spermidine slightly enhanced the reaction (about 10%) (Fig. 3 B, lanes 3-10), a result that suggests that spermidine can assist in the proper folding that is required for generation of a cleavable tRNA molecule, as in the case of hepatitis delta virus RNA (23) . These experiments (Fig. 3 B), in which the native tRNA(CUU) that had been labeled at the 5`-end was used, also showed that no cleavage sites other than p16 were present in the 5` portion of the tRNA. Fig. 3 C shows the effects of temperature on the reaction. In the presence of Mgions, the cleavage reaction proceeded efficiently at 37 °C but not at 10 °C. At 55 °C, the tRNA was nonspecifically degraded.


Figure 3: Determination of the optimal conditions for the cleavage of the native tRNA(CUU) from squid. A, the cleavage reaction was performed in a reaction mixture that contained 30 mM MgCl, 2 mM spermidine, and 50 mM Tris-HCl (pH 7.5) at 37 °C in a final volume of 77 µl. At each time point, 11 µl of the reaction mixture were withdrawn and analyzed. B, the reaction mixture with different concentrations of Mgions was incubated at 37 °C for 3 h with or without spermidine. C, the reaction mixture was incubated for 5 h at the temperatures specified. In each group, the concentration of Mgions was varied. Electrophoresis was performed in 15% polyacrylamide gels that contained 7 M urea and 10% glycerol. The cleavage product is indicated by an arrow in each case.



The Cleavage of tRNA(CUU) Can Be Potentiated by Several Metal Ions

We examined the cleavage of squid tRNA(CUU) in the presence of other metal ions. Both Caand Pbions potentiated cleavage of squid tRNA(CUU) at specific sites (see below). To compare the sites of cleavage in the presence of various metal ions, we incubated 5`-labeled tRNA(CUU) with Mg, Ca, and Pbions separately. In the presence of Caand Mgions, the tRNA was cleaved at the same site. However, the efficiency and specificity of the reaction with Caions were lower than with Mgions (data not shown). By contrast, in the presence of Pb, the tRNA was mainly cleaved at the phosphodiester linkage between Cand G(p18), namely two nucleotides downstream from the cleavage site found with Mgand Caions, confirming the previous data obtained with yeast tRNA(GAA) (data not shown).

Characterization of the End Products of Cleavage in the Presence of Mg, Ca, and PbIons

To characterize the end products of cleavages that occurred in the presence of metal ions, we constructed the pC60K plasmid, which can be transcribed by T7 RNA polymerase to generate the same sequence as that of the native squid tRNA(CUU) with the exception of Uand A. This U-A pair was changed to a G-C pair to allow the plasmid to be transcribed more efficiently (the product was designated tRNA(CUU)(C)).

The plasmid was transcribed in the presence of [-P]UTP, and the resultant labeled transcript was cleaved in the presence of Mgions. The 15-mer cleavage product was isolated by electrophoresis and was further digested with RNase U2 for analysis of the 3` terminus of the oligonucleotide by thin-layer chromatography. Because RNase U2 recognizes purines predominantly, adenosine residues to produce a 2`,3`-cyclic phosphate, this enzyme appears unlikely to modify the 3`-end structure of the 15-mer generated in the presence of Mgions. To our surprise, as shown in Fig. 4A, the major terminus after cleavage was found to be guanosine 3`-phosphate (designated G3`p) and not guanosine 2`,3`-cyclic phosphate (designated G>p), which is a common end product of cleavage in several ribozyme systems (24, 25, 26) . In addition to G3`p, small amounts of G2`p and G>p were also detected. Because RNase U2 cannot cleave a 2`,3`-cyclic phosphodiester bond under the conditions used (as confirmed by the result that an excess of G>p in the reaction mixture could not be cleaved under these conditions; see ``Experimental Procedures''), the result suggests that tRNAwas cleaved intramolecularly in the presence of Mgions at the phosphodiester linkage of p16 with opening of the 2`,3`-cyclic phosphate ring to give rise mainly to G3`p and also to a small amount of G2`p.


Figure 4: The cleavage product generated by Mg ions has mainly a 3`-phosphate and not a 2`,3`-cyclic phosphate ring at its 3`-end. A, analysis of products of digestion with RNase U2 of the 15-mer generated by Mgions by thin-layer chromatography. X and X` are unidentified oligonucleotides produced from internal positions within the 15-mer. B, analysis of products of digestion with nuclease P1 of the same 15-mer by thin-layer chromatography. C, analysis of products of digestion with RNase U2 of 15-mer generated by Caions by thin-layer chromatography. D, analysis of products of digestion with nuclease P1 of the same 15-mer by thin-layer chromatography. E, analysis of products of digestion with nuclease P1 of the 17-mer generated by Pbions by thin-layer chromatography. F, analysis of products of digestion with nuclease P1 of the same 17-mer after treatment of the 17-mer with hydrochloric acid. See ``Experimental Procedures'' for details of these analyses.



To confirm this result, another experiment was performed. The 15-mer cleavage product was digested with nuclease P1 and the digest was analyzed by TLC. As shown in Fig. 4B, a considerable amount of inorganic phosphate was detected in addition to pU derived from internal nucleotides of the oligonucleotide, pG2`p and pG>p. Our interpretation of this result is that the labeled 3`-phosphate in pG3`p that was generated by the cleavage reaction was released by 3`-phosphomonoesterase activity associated with nuclease P1. Because this activity releases the monophosphate of N3`p more efficiently than that of N2`p (3,000-fold preference for N3`p as compared with N2`p; Ref. 27), the presence of a small amount of pG2`p can be explained by this difference in efficiency. The same result was found in the case of the cleavage reaction that occurred in the presence of Caions (Fig. 4, C and D).

To confirm that the Mgions cleave the phosphodiester linkage to form a 2`,3`-cyclic phosphate that is then further opened to the 3`-phosphate, we performed a kinetic experiment as shown in Fig. 5. The amounts of G3`p and G2`p increased with time, whereas the amount of G>p decreased concordantly. The result suggests that the cleavage actually goes through the cyclic intermediate.


Figure 5: Time course of the generation of G>p, G3`p, and G2`p in the cleavage reaction by Mg ions. The experimental conditions are as described in the legend to Fig. 4. At times specified, an aliquot was taken from the reaction tube and was subjected to electrophoresis. The 15-mer was digested with RNase U2, followed by TLC. The radioactivity corresponding to G>p, G3`p, and G2`p was analyzed by an image analyzer (bas2000).



As a control experiment, an end product of cleavage in the presence of Pbions was also analyzed. The pC60K plasmid was transcribed in vitro in the presence of [-P]GTP by T7 RNA polymerase, and the labeled transcript was incubated with Pbions. The resultant 17-mer oligonucleotide was digested with nuclease P1, and the digest was analyzed by TLC as shown in Fig. 4 E. In this case, only pC>p and not Pwas detected, confirming the data obtained in several earlier studies of the cleavage of yeast tRNAin the presence of lead ions. When the 17-mer oligonucleotide was pretreated with hydrochloric acid to open the 2`,3`-cyclic phosphate ring, nuclease P1 released free phosphate groups from pCp (Fig. 4 F), confirming the release of the 3`-phosphate from pGp in the experiment for which results are shown in Fig. 4( B and D). These results unambiguously demonstrated that the cleavage of the tRNA in the presence of Mgions generates mainly a 3`-phosphate terminus and not a 2`,3`-cyclic phosphate ring.

Effect of Con the Cleavage Reaction

Under the optimal ionic conditions, determined as described above (30 mM MgCl, 2 mM spermidine, 50 mM Tris-HCl, pH 7.5) at 37 °C, the efficiency of the cleavage reaction was compared between the tRNA(CUU) and tRNA(UUU), as shown in Fig. 6 . After 8 h of incubation, 48% of tRNA(CUU) had been cleaved, whereas only 3% of tRNA(UUU) had been cleaved. Thus the ratio of the efficiencies of the cleavage reactions was 16 to 1.


Figure 6: Comparison of the efficiency of the cleavage of the two native species of squid tRNAs in the presence of Mg ions. The reaction conditions were the same as described in the legend to Fig. 3 A. The cleavage product is indicated by an arrow.



As shown in Fig. 1, the sequences of the D-loop and D-stem regions of these two tRNAs are the same, suggesting that the T-loop region, which interacts in the tertiary structure with the D-loop, might be responsible for the difference in the efficiencies of the cleavage reactions. In tRNA(CUU) the nucleotide at position 60 is cytidine, whereas in tRNA(UUU) the same position is occupied by uridine. Therefore, we next examined the effect of Con the cleavage reaction by constructing a T7 transcript that contained Uin the sequence background of tRNA(CUU).

The T7 transcript that contained the same sequence as that of tRNA(CUU) with the exception of one base pair, U-A, and U at position 60 in place of C (designated as tRNA(CUU)(U)) was constructed. The efficiency of the cleavage reaction was compared between tRNA(CUU)(C) and tRNA(CUU)(U). Fig. 7 A shows the results of the experiment, in which the 5`-labeled transcript was used. tRNA(CUU)(U) was found to be less effectively cleaved in the presence of Mgions than tRNA(CUU)(C) (about 30% efficiency), indicating that Cmight be responsible for efficient cleavage. Because tRNA(CUU)(U) was cleaved to some extent (Fig. 7 A, lanes 10-11), the nucleotide at position 60 is not the sole constituent required for cleavage. A similar result was obtained in the case of the 3`-labeled transcript, as shown in Fig. 7 B. In both experiments, each transcript was found to be cleaved at a rate severalfold lower than that of the respective native tRNA. Several additional cleavages other than that between Gand Dwere observed (these additional cleavages occurred at the anticodon loop) (data not shown). However, because the additional products were also detected in control assays in the absence of Mgions (Fig. 7, A, lanes 6 and 12 and B, lanes 6 and 12), incubation at 37 °C was presumed to degrade the transcripts. Because both transcripts could be aminoacylated to the same extent by using an S-100 extract of bovine liver (data not shown), they may remain the native conformation of tRNA molecule. These results suggest that the stable tertiary structure of the native tRNA(CUU) molecule might be responsible for the highly specific and efficient cleavage that occurs in the presence of Mgions, although there remains the possibility that the alteration of a nucleotide pair of U-Ato G-Cis responsible for this change of the specificity and efficiency of the reaction.


Figure 7: Comparison of the efficiency and specificity of the cleavage of tRNA(CUU)(C) and tRNA(CUU)(U). The two tRNAs were labeled at the 5` terminus ( A) or at the 3` terminus ( B) and used as substrates for the cleavage reaction. The cleavage products, namely the 15-mer ( A) and the 61-mer ( B), are indicated by arrows. The reaction conditions were the same as described in the legend to Fig. 3 A. In the case of lanes 6 and 12, the reactions were performed for 5 h in the absence of Mgions and in the presence of 1 mM EDTA. Electrophoresis was performed in 15% polyacrylamide gels that contained 7 M urea and 10% glycerol for 1 h.



Integrity of the tRNA Molecule May Be Required for Cleavage in the Presence of MgIons

In order to investigate the structural constraints required for the cleavage reaction, native tRNA(CUU) labeled at the 5` or the 3` terminus was partially hydrolyzed with alkali. The hydrolysates were separated by electrophoresis on a polyacrylamide gel, and from each ladder the partially hydrolyzed tRNA molecules were extracted. Each molecule lacking a part of the tRNA sequence was incubated under the conditions of the cleavage reaction for 3 h, as described in the legend to Fig. 3 A. Fig. 8A shows the results of the reactions with, as substrates, the hydrolysates of tRNA(CUU) labeled at its 5`-end by [-P]ATP. The intact tRNA(CUU) of 76 nucleotides that was extracted from the denaturing gel was a good substrate for the cleavage reaction (Fig. 8 A, lane 1), indicating that the denaturation and renaturation procedures in this experiment did not substantially change the specificity and efficiency of the cleavage, as compared with the cleavage of the initially purified tRNA(CUU). As shown in lane 2 of Fig. 8A, the specificity and efficiency of the cleavage were severely disrupted in the case of tRNA(CUU) with the one-nucleotide deletion of A at the 3`-end. With progressive loss of the 3` portion of the tRNA, a new cleavage site emerged in the extra loop region. After deletion of several nucleotides, the specific cleavage at the position of p16 tended to disappear. In the case of the tRNA(CUU) labeled with [5`-P]pCp at the 3`-end, a similar result was obtained (Fig. 8 B). These results suggest that intact tRNA(CUU) is a prerequisite for the specificity and efficiency of the cleavage between Gand D.


Figure 8: Integrity of conformation of native tRNA(CUU) is required for the efficient cleavage. A and B, analyses of the cleavage of partially hydrolyzed tRNA molecules that lacked a part of the sequence and had been labeled at the 5` terminus ( A) and the 3` terminus ( B), respectively. Electrophoresis was performed in 15% polyacrylamide gels that contained 7 M urea and 10% glycerol. The product of cleavage at a specific site (p16) is indicated by an arrow. The number above each lane indicates the length of the fragment in that lane. Lane M shows an RNase T1 ladder. Lane C shows a labeled RNA sample without treatment of alkali.




DISCUSSION

Highly Specific and Efficient Cleavage of Squid tRNAin the Presence of MgIons

In this report, we have demonstrated that Mgions promote the cleavage of squid tRNA. As described in the Introduction, the specific cleavage of yeast tRNAbetween Dand Gin the presence of Pbions has been studied extensively as a model for the role of metal ions during the chemical step of RNA catalysis (2, 3) . The cleavage is highly efficient and specific for this site, providing a good tool for deduction of the tertiary structures of tRNA by the analysis of the positions of metal ions in tRNAs (4, 7, 28) . In addition to Pbions, several other ions, such as Znand Euions, are capable of potentiating the cleavage of purified tRNAs at precise locations (5, 6, 7) . Mgions can also potentiate reactions of purified tRNAs, such as elongator tRNAand tRNAof yeast and lupin (7, 28, 29) . However, in these cases, the efficiency and specificity of the cleavage in the presence of Mgions are not as great as in the case presented here, and all of the Mg-promoted cleavage reactions reported to date occur only under alkaline conditions (pH 8.5-9.5). In our experiments, about 50% of substrates were cleaved in 8 h, and, moreover, the cleavage occurred not only in the presence of Mgions but also in the presence of Caions at neutral pH. Accordingly, the cleavage reaction presented here is unique in that a tRNA other than tRNAcan be cleaved in the presence of Caions or Mgions at neutral pH.

A Cytidine Residue at Position 60 Is Required for Efficient Cleavage

An analysis of the cleavage of the yeast tRNAin the presence of Pbions was made using the T7 RNA polymerase-generated transcript with the same sequence as that of the native molecule (30) . The rate of cleavage of this transcript was about half that of the native tRNA. Furthermore, mutations that disrupted tertiary interactions between the T- and D-loops reduced the cleavage rate, and some mutants, in particular UC, in which Uwas replaced by C, or CU, in which Cwas replaced by U, reduced the cleavage rate 10-fold (30) . Crystallographic data indicate that three Pbions bind to tRNAand that one particular Pbion (designated Pb(1) ) binds tightly to Uand Cin the T-loop (2, 3) . Thus, it seems that the tertiary interactions of tRNA are important both in positioning the lead-coordinating nucleotides of Uand Cand in keeping the 2`-hydroxyl of the substrate near the Pbion. In the present study, we determined the nucleotide sequences of two isoacceptor tRNAs. tRNA(CUU) was cleaved very efficiently in the presence of Mgions, whereas tRNA(UUU) was not cleaved. The influence of Cin tRNA(CUU) on the cleavage reaction was confirmed by the experiment with T7 RNA polymerase-generated transcripts that contained Cand U, respectively, with the sequence background of squid tRNA(CUU) (Fig. 7). In yeast tRNA, Mg-binding sites were identified by x-ray crystallography (31, 32, 33, 34, 35) . One of the Mgbinding sites (designated Mg (3) ) makes contact with C, and, therefore, our single-base substitution is likely to have changed the position or the affinity of an Mgion in such a way that the cleavage was less efficient.

In another experiment, as shown in Fig. 8, the cleavage activity was disrupted by a one-nucleotide deletion from the 5`- or 3`-end, respectively, of squid tRNA(CUU). Recently, Limmer et al. (36) reported that the single-stranded 3`-terminal end (5`-NCCA-3`) of tRNA influences the structure and stability of the acceptor stem via an effect on stacking. Therefore, it is likely that our deletion mutants had lost the stability of their acceptor stems, with subsequent destabilization of the entire tRNA structure that is required for the specific and efficient cleavage of squid tRNA(CUU) in the presence of Mgions.

The Predominant Product of Cleavage Terminates with a 3`-Phosphomonoester

The most remarkable finding of the present study was that the cleavage product terminated mainly with a 3`-terminal phosphate and not with a 2`,3`-cyclic phosphodiester. Except for a simple example reported by Pan and Uhlenbeck (Ref. 16; see Introduction), all ribozymes, such as hammerhead, hairpin, and hepatitis delta ribozymes, seem to be unable to catalyze hydrolysis of the 2`,3`-cyclic phosphate produced from the transesterification reaction (24, 25, 26) . By contrast, self-splicing group I and group II introns and the RNA subunit of ribonuclease P catalyze the attack of an exogenous nucleophile, yielding 3`-OH and 5`-phosphate groups (8, 9, 37) . Our finding is not the first example of cleavage of a tRNA in the presence of Mgions. However, because the structure of the end product has not previously been rigorously examined, it is unclear whether the 3`-terminal phosphate structure is generated specifically in the case of squid tRNA(CUU) or is a general feature of such cleavage of all tRNAs in the presence of Mgions.

Pan and Uhlenbeck (16) reported a particular variant of the ``leadzyme'' that not only catalyzes the cleavage of a phosphodiester bond at a specific site but also promotes the hydrolysis of 2`,3`-cyclic phosphate to produce specifically a 3`-monophosphate. Our present findings provide the second example of such a two-step reaction. These two examples seem to be unique to date, but they have features in common with reactions catalyzed by many protein ribonucleases. Whereas the ``leadzyme'' is a product of in vitro selection experiments, squid tRNAis not an artificial product but a native molecule that exists in vivo. It is of interest, also, that the two-step cleavage of squid tRNAis catalyzed by biologically ubiquitous ions such as Mgand Caat neutral pH and not by toxic, nonessential cations such as Pb.

It is becoming evident that the transesterification reaction involved in the self-splicing in group I or II introns and possibly in the splicing of pre-mRNA is really catalyzed by metal ions such as Mg. The RNAs themselves are not involved directly in the chemistry of the catalysis but are necessary to maintain a specific tertiary structure that binds metal ions within a precise configuration (10, 38, 39) . The cleavage of squid tRNA(CUU) described herein required the entire tRNA structure for effective cleavage in the presence of Mgions. The finding that an Mgion situated within a certain configuration can cleave RNA in the same way as present day ribonucleases suggests various roles for metal ions during evolution in the RNA world (40) .


FOOTNOTES

*
This work was supported by a grant-in-aid for specially promoted research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The tRNA sequences reported in the present study will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence data bases with the accession numbers D45190 and D45191.

§
To whom correspondence should be addressed. Tel. and Fax: 81-45-923-1136.

M. Matsuo, Y. Abe, Y. Saruta, and N. Okada, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. T. Ueda and H. Himeno for useful discussions and C. Takemoto for providing the aminoacyl-tRNA synthetase.


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