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Originally published In Press as doi:10.1074/jbc.M202023200 on March 28, 2002

J. Biol. Chem., Vol. 277, Issue 23, 20510-20517, June 7, 2002
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Plasticity of Recognition of the 3'-End of Mischarged tRNA by Class I Aminoacyl-tRNA Synthetases*

Brian E. NordinDagger and Paul Schimmel§

From The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, California 92037

Received for publication, February 28, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Certain aminoacyl-tRNA synthetases prevent potential errors in protein synthesis through deacylation of mischarged tRNAs. For example, the close homologs isoleucyl-tRNA synthetase (IleRS) and valyl-tRNA synthetase (ValRS) deacylate Val-tRNAIle and Thr-tRNAVal, respectively. Here we examined the chemical requirements at the 3'-end of the tRNA for these hydrolysis reactions. Single atom substitutions at the 2'- and 3'-hydroxyls of a variety of mischarged RNAs revealed that, while acylation is at the 2'-OH for both enzymes, IleRS catalyzes deacylation specifically from the 3'-OH and not from the 2'-OH. In contrast, ValRS can deacylate non-cognate amino acids from the 2'-OH. Moreover, for IleRS the specificity for a 3'-O location of the scissile ester bond could be forced to the 2'-position by introduction of a 3'-O-methyl moiety. Cumulatively, these and other results suggest that the editing sites of these class I aminoacyl-tRNA synthetases have a degree of inherent plasticity for substrate recognition. The ability to adapt to subtle differences in mischarged RNAs may be important for the high accuracy of aminoacylation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The genetic code is based on the accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetases (1, 2). These enzymes synthesize aminoacyl-tRNA in two steps. The amino acid is first reacted with ATP to give an activated aminoacyl adenylate, and then transesterified to the 3'-end of the tRNA. Aminoacyl-tRNA synthetases must precisely recognize both amino acid and tRNA substrates to yield the correct product. While the structural diversity of tRNA molecules allows for rigorous selection based on RNA-protein interactions, differentiating between closely related amino acids is more challenging. Years ago, Pauling (3) noted the intrinsic difficulty for isoleucyl-tRNA synthetase in the recognition of isoleucine over valine through simple binding interactions.

Valine, which differs from isoleucine by a single methylene unit, is activated by Escherichia coli IleRS1 only 180-fold less efficiently than isoleucine (4). However, the substitution of valine for isoleucine at isoleucine codons in the cell is less than 1 in 3000 (5). The increased specificity is a result of the RNA-dependent editing of misactivated valine by IleRS (6, 7). A highly related class I aminoacyl-tRNA synthetase, valyl-tRNA synthetase, faces a similar dilemma in the accurate aminoacylation of tRNAVal. Threonine, an isostere of valine, is activated at a rate 250-fold reduced from that of valine (8). Like IleRS, ValRS prevents the misincorporation of threonine into proteins through the RNA-dependent editing of misactivated threonine (9).

These reactions strictly require the presence of the cognate tRNA (6, 10). In the absence of tRNA, the enzymatically generated misactivated adenylates remain in the active site, sequestered from hydrolysis. Upon addition of cognate tRNA the misactivated amino acids are hydrolyzed, regenerating the free tRNA and amino acid, while converting 1 equivalent of ATP to AMP. A prominent mechanism for editing misactivated amino acids is the rapid hydrolysis of transiently mischarged tRNA (7, 9). This reaction is catalyzed at a second active site on IleRS and ValRS. This site is located within a large insertion (termed CP1) into the canonical class I aminoacyl-tRNA synthetase active-site fold. The CP1 domain as an isolated polypeptide hydrolyzes its cognate mischarged tRNA (11). Crystallographic analysis of Thermus thermophilus IleRS pinpointed the editing site to a pocket of essentially invariant amino acids within CP1 located ~30 Å from the aminoacylation active site (12). This site binds valine but sterically excludes isoleucine. A co-crystal structure of Staphylococcus aureus IleRS with tRNAIle suggested how the tRNA may place its 3'-end in the editing site (13). However, specific interactions in the editing site could not be observed. More recently, the co-crystal structure of T. thermophilus ValRS bound to tRNAVal demonstrated a similar mode of tRNA binding (14). Although a mischarged amino acid could be modeled at the end of the tRNA, neither this model nor mutational analysis established a hydrolytic mechanism (15, 16).

Early work demonstrated an important role for the 3'-end of tRNAIle and tRNAVal in editing (17-19). However, the exact nature of this role was not determined. Model studies of uncatalyzed ester hydrolysis demonstrated that a cis-hydroxyl stimulates hydrolysis, likely via intramolecular hydrogen bonding (20). While its effect may be important for the efficiency of deacylation, the importance of the terminal hydroxyls could be related to the rapid transacylation known to occur between the two cis-hydroxyls (with a rate of ~10 s-1) (21). Thus, while aminoacylation is specific to a particular hydroxyl (2'-OH in the cases of IleRS and ValRS (22)) deacylation could potentially occur from either the 2'- or 3'-OH group.

To address issues that could not be taken up in earlier work because of then existing technical limitations, we constructed a variety of mischarged RNA substrates having different substitutions at the positions of the terminal hydroxyl groups. Using these substrates we were able to test directly whether deacylation occurred from a specific hydroxyl, whether transacylation from the 2'- to the 3'-OH was required for deacylation, and finally, whether a clear chemical role for vicinal hydroxyls could be identified.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein Expression and Purification-- Wild-type E. coli IleRS was overexpressed in E. coli strain MV1184 from plasmid pKS21, which contains the gene for IleRS under control of the lac promoter (23). The T242P mutant of E. coli IleRS was overexpressed in E. coli strain PS2766 (Delta ilv, Delta ileS203::kan+) from plasmid pVDC434, a derivative of pBAD18 containing the gene for T242P IleRS under the control of an arabinose-inducible promoter (24). Purification of the wild-type and mutant IleRS was as described previously (25). IleRS concentrations were determined by active site titration (26).

Wild-type E. coli ValRS was overexpressed as a C-terminal His6-tagged protein in E. coli strain BL21(DE3) from a pET-21b (Novagen, Madison, WI) derivative containing the gene for ValRS (27). Standard protocols were used for purification. The T222P mutant of E. coli ValRS was overexpressed in E. coli strain PS2801 (Delta valS::kan+) from plasmid pVDC447, a pBAD18 derivative containing the gene for T222P ValRS (28). Purification was essentially identical to the protocol used for IleRS. Wild-type and the T379A,T380A double mutant (these mutations are in the editing site and do not affect aminoacylation activity with valine) yeast (Saccharomyces cerevisiae) ValRS were overexpressed as C-terminal His6-tagged proteins in the yeast strain CW1, which has the chromosomal copy of ValRS deleted.2 Both proteins were purified using standard methods for His6-tagged proteins. ValRS concentrations were determined by the Bradford dye-binding assay.

The plasmid pET-22-CCA, which encodes a C-terminal His6-tagged E. coli tRNA nucleotidyltransferase (tRNA NTase), was a generous gift from the laboratory of Nancy Maizels and Alan Weiner (29). This plasmid was transformed into E. coli BL21(DE3) cells and the resulting strain was used for overexpression and purification. Protein concentration was determined by the Bradford assay.

RNA Substrates-- Mature E. coli tRNAIle (GAU) was isolated from E. coli strain MV1184 containing the plasmid pES300, which allows for the isopropyl-1-thio-beta -D-galactopyranoside-inducible overexpression of tRNAIle (30). Purification was as described previously (31). The resulting preparations of tRNAIle were generally comprised of 50-70% tRNAIle with the remainder being other cellular tRNAs. Transcripts of tRNAIle missing the terminal A76 nucleotide (Delta A76 tRNAIle) were found to be superior to full-length transcripts for 3'-end modification using tRNA NTase. The plasmid pBNt02 was constructed for use as a template for Delta A76 tRNAIle transcription in vitro using T7 RNA polymerase. A small segment of DNA was inserted into plasmid pMF15 (32) to place a FokI recognition site, such that cleavage with FokI generated a template strand terminating with C75. Linearization with FokI was done at 37 °C for 2 h at an enzyme concentration of 0.3 units/µg of DNA. Transcription reactions and purification were as described previously (33). Mature tRNAVal was purchased from Sigma. Concentrations of tRNA preparations were determined by measuring the plateau level of charging with cognate amino acid.

RNA minihelices were synthesized on an Expedite 8909 synthesizer (PE Biosystems, Foster City, CA) and deprotected as described (34). Minihelices with modified 3'-ends were synthesized directly from the corresponding modified nucleoside CPG resins (ChemGenes, Boston, MA). Following deprotection the minihelices were purified on a DNAPac PA-100 ion exchange high performance liquid chromatography column (Dionex, Sunnyvale, CA). Minihelix concentrations were determined from absorbance at 260 nm.

tRNA 3'-End Modification Reactions-- The tRNA NTase-catalyzed pyrophosphorolysis and nucleotide exchange procedure developed in the Hecht laboratory was used exclusively for the preparation of tRNAs with modified 3'-terminal bases (35). The only significant change to their procedure was use of E. coli tRNA NTase, instead of the yeast enzyme. The exchange reaction was performed in 20 mM Tris-HCl (pH 8.5), 20 mM MgCl2, 1 mM sodium pyrophosphate, 6 mM ATP (or analog), 15 µM tRNA, and 5 µM tRNA NTase. In all assays where A76 tRNA is reported as the substrate, the tRNA was put through the tRNA NTase exchange procedure using ATP to regenerate the wild-type 3'-end. The 3'-deoxy-ATP (3'-dATP) was purchased from Sigma. The 2'-fluoro- and 3'-fluoro-ATP were purchased from Purimex (Göttingen, Germany). The reaction was allowed to proceed for 4 h at 37 °C and then ethanol precipitated, resuspended, and the product purified on a 10% polyacrylamide, 8 M urea gel. Final products were generally found to be between 98 and 99% modified as judged by aminoacylation assays. The nucleotide exchange procedure was found to work well with the Delta A76 tRNAIle transcript, and found to be necessary (as opposed to a simple addition reaction) due to an approximate 10% contamination of Delta A76 tRNAIle with full-length transcript, likely from improper T7 RNA polymerase termination. Concentrations of 3'-end modified tRNAs were determined (where possible) from their plateau levels of aminoacylation with cognate amino acid.

Aminoacylation Assays-- The aminoacylation of RNA substrates was determined by measuring the trichloroacetic acid precipitable radioactivity generated via the attachment of tritiated amino acid to RNA (25). The assays were performed at room temperature in 20 mM HEPES (pH 7.5), 150 mM NH4Cl, 100 µM EDTA, 10 mM MgCl2, 2 mM ATP, and 10 nM inorganic pyrophosphatase. Assays with IleRS used 500 nM enzyme, 2 µM tRNA, and 15 µM of either [3H]isoleucine (1.67 mCi/µmol) or [3H]valine (1.65 mCi/µmol). Assays with ValRS used 1 µM enzyme, 5 µM tRNA, and 15 µM of either [3H]valine (1.65 mCi/µmol) or [3H]threonine (1.65 mCi/µmol). To demonstrate stoichiometric mischarging of 3'-dA76 tRNAVal, 5 µM ValRS and 1 µM tRNA was used.

Mischarging Reactions-- Mischarged RNA substrates for IleRS were prepared using two primary techniques. Because IleRS and ValRS both charge the 2'-OH, producing mischarged RNA substrates that have altered 2'-OH groups (i.e. 2'-dA) is challenging. A search for reaction conditions where this mischarging could take place revealed that, under conditions of no monovalent cations and 30% dimethyl sulfoxide (Me2SO), yeast ValRS had a low, but easily detectable mischarging activity toward 2'-dA76 tRNAIle. Additionally, it was found that the T379A,T380A double mutant yeast ValRS had an equivalent activity under these conditions. (Larger quantities of the mutant ValRS were available to use in mischarging preparations.) Preparative mischarging reaction conditions were 25 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 30% Me2SO, 1 mM ATP, 10 nM inorganic pyrophosphatase, 5 µM [3H]valine (23 mCi/µmol), 500 nM yeast ValRS (or double mutant), and 5 µM tRNA or 100 µM minihelix. Reactions were incubated at room temperature for 4-6 h, then extracted with phenol/chloroform (1:1) twice, ethanol precipitated, and resuspended in 10 mM sodium acetate (pH 4.5). This method was used for mischarging tRNAIle transcripts, minihelices, and their respective 3'-end variants. By using transcripts and synthetic RNAs it could be guaranteed there was no trace contamination of tRNAIle and minihelixIle substrates with tRNAVal species.

For the preparative mischarging of mature tRNAIle and the related 3'-end variants, an enzyme that has no cross-reactivity with any potential remaining tRNAVal in the tRNA samples was required. This requirement was met by using an editing-deficient mutant (T242P) (16) of E. coli IleRS under standard conditions where tRNA recognition is intact. Mischarging reactions were performed in 20 mM HEPES (pH 7.5), 100 µM EDTA, 150 mM NH4Cl, 10 mM MgCl2, 1 mM ATP, 10 nM inorganic pyrophosphatase, and 5-10 µM [3H]valine (23 mCi/µmol), with 5 µM tRNA substrate and 20 µM T242P IleRS. Mischarging reactions were incubated at room temperature for 30-60 min and worked up as described above.

The same strategy was used to prepare mischarged tRNAVal and the respective 3'-end variants. The T222P mutant of E. coli ValRS was found to effectively mischarge wild-type and other tRNAVal derivatives with threonine (28). The conditions for mischarging were identical to those for the T242P IleRS mischarging of tRNAIle, except that 8 µM [3H]threonine (50 mCi/µmol) replaced valine. T222P ValRS was used at a concentration of 5 µM, as was the tRNA substrate. The yield of mischarged product depended on the incubation time more critically than seen with tRNAIle mischarging reactions, likely due to a low level of editing activity with T222P ValRS. Optimal yields were achieved using a 5-min incubation for mischarging wild-type tRNAVal, a 20-30-min incubation with 3'-dA76 tRNAVal, and a 1-h incubation with 3'-fluoro-A76 tRNAVal. All reactions were performed at room temperature and worked up as described above.

Deacylation Assays-- Deacylation was also measured using the trichloroacetic acid precipitation technique. Deacylation of mischarged mature tRNAIle was done at room temperature in 150 mM Tris-HCl (pH 7.5), 20 mM MgCl2, using 1 µM mischarged tRNA and 50 nM IleRS. For comparisons of the deacylation activity of Val-2'-dA76 tRNAIle with other tRNAs, tRNAIle transcripts were used. Not only were transcripts free of tRNAVal contamination, but the 3'-end-modified derivatives also had less contamination of their 3'-ends with residual A76 because the exchange reactions utilized Delta A76 tRNAIle as the source tRNA (generally 99.5% exchange was achieved as opposed to 98-99%). The deacylation assays using these mischarged transcript-derived substrates were performed under the same conditions as those conditions for mature tRNAIle (isolated from cells) and its 3'-end variants, except 600 nM mischarged tRNA and 200 nM IleRS were used. ValRS has a slightly higher deacylation activity, so assays measuring the deacylation of mischarged tRNAVal and its derivatives were done with 10 nM ValRS and 1 µM mischarged tRNA. Deacylation assays using mischarged minihelices were also done with 600 nM mischarged minihelix and 200 nM IleRS. Deacylation of Val-3'-dA76 tRNAIle was achieved using very high enzyme concentrations under conditions where substrate recognition is altered. Both mischarged and correctly charged 3'-dA76 tRNAIle (1 µM) were deacylated in 150 mM Tris-HCl (pH 8.5), 20 mM MgCl2, 20% methanol, using 2 µM IleRS.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IleRS Mischarges tRNA Substrates with Altered 3'-Ends-- Early work established that IleRS and ValRS utilize the 2'-OH as the initial site of aminoacylation, a feature now known to be characteristic of all class I aminoacyl-tRNA synthetases (22, 36, 37). To further investigate the role of the 3'-end of tRNA in determining the specificity and mechanism of the deacylation reaction, a variety of substrates with altered 3'-ends were constructed. Replacement of the 3'-OH group of tRNAIle by a hydrogen atom (giving 3'-dA76 tRNAIle) yields a tRNA that is devoid of editing activity (Fig. 1A) (17). As expected, this modification does not significantly affect its aminoacylation activity relative to A76 tRNAIle (Fig. 1B). The combination of high aminoacylation activity and lack of editing manifests itself in 3'-dA76 tRNAIle being completely mischarged with valine by IleRS. This mischarging is in striking contrast to the cis-diol containing A76 tRNAIle, which is not detectably mischarged with valine.


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  		Fig. 1.   Aminoacylation of tRNAIle 3'-end variants. A, mischarging with valine. IleRS does not mischarge A76 tRNAIle. In contrast, those substrates with a modified 3'-OH are completely mischarged with valine. B, aminoacylation with isoleucine. An intact 2'-OH is required for proper aminoacylation by IleRS and thus, the 2'-dA76 and 2'-fluoro-A76 tRNAIle (2'F-A76) substrates are not aminoacylated. The A76, 3'-dA76, and 3'-fluoro-A76 tRNAIle (3'F-A76) substrates are all aminoacylated to similar levels.

To more closely examine the properties of the 3'-OH group of tRNAIle that are critical for editing, a 3'-fluoro-A76 tRNAIle was constructed. Fluorine better emulates the electronegative properties of the OH group and may even serve as a hydrogen bond acceptor (38-40). If these were the properties of the OH group that promoted editing, then 3'-fluoro-A76 tRNAIle should be difficult to mischarge. However, IleRS rapidly mischarged 3'-fluoro-A76 tRNAIle with valine (Fig. 1A). As expected, we observed no charging or mischarging with 2'-dA76 tRNAIle or 2'-fluoro-A76 tRNAIle.

It is worth noting that the data in Fig. 1 also demonstrate the purity of the tRNA products isolated from the tRNA NTase exchange reactions. As the relative charging plateaus (isoleucine versus valine) for 3'-dA76 tRNAIle and 3'-fluoro-A76 tRNAIle are equal for both tRNA substrates, it is likely that virtually no wild-type tRNAIle (which only charges with isoleucine) remains in the preparations. The same conclusion can be drawn from the observation that neither 2'-dA76 tRNAIle nor 2'-fluoro-A76 tRNAIle display any charging activity.

The Terminal 3'-OH of tRNAIle Is Important for Deacylation of Mischarged tRNAIle-- IleRS has a potent deacylation activity toward mischarged A76 tRNAIle (Fig. 2A). This activity is completely abolished by the replacement of the terminal 3'-OH with either a hydrogen or fluorine atom. (While the plots in Fig. 2A show a slow deacylation rate for Val-3'-dA76 tRNAIle and Val-3'-fluoro-A76 tRNAIle, under these conditions the rates were identical to the rates in the absence of enzyme.) Under conditions designed to promote rapid deacylation rates (4 µM mischarged tRNA substrate, 1 µM IleRS), the complete lack of deacylation activity toward Val-3'-dA76 tRNAIle is striking (Fig. 2B). The A76 tRNA was completely deacylated in 2-3 min, whereas the 3'-dA76 tRNAIle was only about 3% deacylated after 1 h. This amounts to a more than 750-fold decrease in deacylation rate.


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  		Fig. 2.   Deacylation of mischarged tRNAIle variants. A, mischarged A76 tRNA is deacylated rapidly, while those tRNAs without an intact 3'-OH show essentially no deacylation. B, after an hour incubation of 4 µM mischarged 3'-dA76 tRNAIle with 1 µM IleRS it is still nearly 100% aminoacylated. In contrast, under the same conditions, mischarged A76 tRNAIle is almost completely deacylated within 2 min. The rate difference is at least 750-fold, corresponding to a minimum energy difference of about 4 kcal/mol. Val-3'F-A76, valyl-3'-fluoro A76.

IleRS Deacylates Mischarged 2'-dA76 Substrates-- The results with 3'-dA76 tRNAIle and 3'-fluoro-A76 tRNAIle confirm and expand upon previous work detailing the importance of the 3'-OH group in hydrolytic editing. The question still remains as to what is the mechanistic reason behind the importance of the 3'-OH. One possibility is that the 3'-OH serves a catalytic function to facilitate deacylation from the 2'-OH. Alternatively, the mischarged amino acid may be esterified to the 3'-OH just prior to hydrolysis. The well characterized 2'- to 3'-transacylation reaction on the cis-diol-containing wild-type tRNA could reposition the amino acid. A similar transacylation is not possible for either 3'-dA76 or 3'-fluoro-A76 tRNAIle. If transacylation were a prerequisite to deacylation, then a mischarged 2'-dA76 tRNAIle (containing a 3'-aminoacyl linkage) might be expected to be a substrate for deacylation.

To investigate this possibility we had to develop a method for producing mischarged 2'-dA76 substrates. We attempted to mischarge 2'-dA76 substrates using a number of different aminoacyl-tRNA synthetases and reaction conditions. For this purpose, we used both transcripts (as opposed to the mature, cell-isolated tRNA used in the previous experiments) of tRNAIle and RNA minihelices based on the acceptor-TPsi C stem of tRNAIle (Fig. 3). (IleRS, like many aminoacyl-tRNA synthetases, specifically charges minihelices based on its cognate tRNA (41, 42).) Most mischarging trial reactions were unsuccessful. Neither editing-deficient mutants of IleRS nor E. coli or Bacillus stearothermophilus ValRS had any charging activity with 2'-dA76 substrates. The highest degree of mischarging was achieved using ValRS from yeast. While the reactions were not efficient, about 6-8% of the input 2'-dA76 tRNAIle and 1% of 2'-dA76 minihelixIle was mischarged in incubations using yeast ValRS in the presence of 30% dimethyl sulfoxide (Me2SO) and no monovalent cations. The T379A,T380A double mutant yeast ValRS gave identical yields and was used interchangeably with wild-type yeast ValRS (see "Experimental Procedures").


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  		Fig. 3.   RNA substrates for IleRS. The tRNAIle molecule (left) is comprised of two helical domains that fold into an L-shape. The upper, acceptor domain is formed by the stacking of the acceptor stem on TPsi C-stem-loop. The D-stem-loop stacks on the anticodon stem-loop to create the lower domain. MinihelixIle (right) is a simple RNA hairpin that mimics the acceptor domain of tRNAIle. IleRS specifically recognizes and charges minihelixIle relative to minihelices of non-cognate sequences (41, 48).

Because the fraction of RNA that was successfully mischarged in these preparations was low, contamination of starting materials with wild-type (non-exchanged) RNAs was investigated. The 2'-dA76 tRNAIle used in these reactions was derived from Delta A76 tRNAIle transcripts. This starting material has the advantage of being free of any tRNAVal species (that might be recognized by ValRS) and of having the lowest possible contamination with the cis-diol containing A76 tRNAIle that can arise from incomplete exchange of the A76 terminus. Under standard aminoacylation conditions with isoleucine, 2'-dA76 tRNAIle transcripts showed about 0.5% of the charging level that would be expected based on its absorbance at 260 nM. However, the large excess of 2'-dA76 tRNAIle in these preparations could be inhibiting the aminoacylation of A76 tRNAIle and thus masking the amount of A76 tRNAIle present. To rule out this possibility, a control compared the level of aminoacylation of 2'-dA76 tRNAIle alone to that of 2'-dA76 tRNAIle intentionally spiked with 3% A76 tRNAIle. The reaction containing the 3% A76 tRNAIle showed complete charging in 10 min (data not shown), while the 2'-dA76 tRNAIle alone showed almost no charging. Thus, the 6-8% yield of mischarged 2'-dA76 tRNAIle cannot be due to contamination with A76 tRNAIle. A similar experiment showed that 2'-dA76 minihelixIle was free of wild-type minihelixIle. This result was expected, as the starting material for chemical RNA synthesis is homogenous.

These mischarged 2'-dA76 substrates were tested in deacylation assays relative to their A76 and 3'-dA76 variants. Both Val-2'-dA76 tRNAIle and Val-2'-dA76 minihelixIle were efficiently deacylated by IleRS. Relative to Val-A76 tRNAIle, the 2'-dA76 derivative showed an approximate 5-fold decrease in the initial rate of deacylation (Fig. 4A). This decrease is in striking contrast to the 750-fold or more drop in deacylation rate observed with Val-3'-dA76 tRNAIle. The hydroxyl specificity of deacylation was maintained with regards to the minihelix, as a Val-3'-dA76 minihelixIle was completely resistant to hydrolysis (Fig. 4B). No difference in rate was detected between the deacylation rates for Val-minihelixIle and Val-2'-dA76 minihelixIle.


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  		Fig. 4.   IleRS deacylates mischarged 2'-dA76 RNA substrates. A, deacylation of mischarged tRNAs. The 2'-dA76 tRNAIle is deacylated by IleRS, although at a reduced rate relative to A76 tRNAIle. B, deacylation of mischarged minihelices. Minihelices based on the acceptor/TPsi C stem of tRNAIle are specifically deacylated by IleRS at a somewhat slower rate than are the full tRNAs. The hydroxyl specificity is the same as seen with the full tRNAs.

The 3'-OH of tRNAVal Is Important for Preventing Misacylation by ValRS-- To compare how editing specificity may have adapted through evolution, we performed analogous experiments with the closely related ValRS. While, no misacylation of A76 tRNAVal was detected in any of the conditions tested, substitution of the 3'-OH group of tRNAVal with either a hydrogen or fluorine atom gave a tRNA that is mischarged with threonine (Fig 5A). Additionally, these modifications had little effect on the aminoacylation activity with valine (Fig. 5B). Under the assay conditions used (Fig. 5, A and B), the plateau level of charging of 3'-dA76 tRNAVal with threonine was significantly lower than with valine. When aminoacylation reactions were performed with higher concentrations of ValRS, the charging plateaus with valine and threonine were identical for 3'-dA76 tRNAVal (Fig. 5C). (Thus, the nucleotide exchange reactions with 3'-dATP gave essentially pure 3'-dA76 tRNAVal, and the observed intermediate threonine mischarging plateau (Fig. 5A) cannot be due to contamination with A76 tRNAVal.) Mischarging of 3'-fluoro-A76 tRNAVal with threonine was complete even under the conditions of a lower ValRS/tRNA ratio than needed for complete mischarging of 3'-dA76 tRNAVal (Fig. 5A).


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  		Fig. 5.   Aminoacylation of tRNAVal 3'-end variants. A, aminoacylation with threonine. A76 tRNAVal is not mischarged with threonine by ValRS. Under these conditions the 3'-dA76 tRNAVal is mischarged to ~50% completion, while 3'-fluoro-A76 tRNAVal (3'F-A76) is essentially 100% mischarged. B, aminoacylation with valine. Like IleRS, ValRS initially charges the 2'-OH. Those tRNA substrates with a modified 2'-OH are not aminoacylated, while those with an intact 2'-OH are efficiently charged. C, stoichiometric mischarging of 3'-dA76 tRNAVal. At high ValRS/tRNA ratios the level of threonine incorporation into 3'-dA76 tRNAVal matches the level of valine incorporation. This demonstrates that the 3'-end is completely modified with the 3'-deoxy analog. 2'F-A76, 2'-fluoro-A76.

ValRS Does Not Require an Intact 3'-OH to Edit Mischarged tRNA-- In contrast to IleRS, a mischarged 3'-dA76 substrate is a substrate for deacylation (Fig. 6A). (A similar finding was reported for yeast ValRS (19).) This modification decreases the editing efficiency of both IleRS and ValRS, yet the observed ValRS catalyzed deacylation of Thr-3'-dA76 tRNAVal demonstrates a marked difference in substrate specificity for editing. The mischarging with threonine of 3'-dA76 tRNAVal is the result of competing aminoacylation and deacylation reactions (Fig. 5A). Because the deacylation activity is reduced it cannot keep pace with the mischarging.


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  		Fig. 6.   Deacylation of mischarged tRNAVal variants. A, using 10 nM ValRS, mischarged tRNAVal is rapidly deacylated. Unlike IleRS, ValRS shows deacylation activity toward a mischarged 3'-dA76 tRNA. The deacylation rate for this substrate is reduced relative to A76 tRNAVal. Under these conditions, the deacylation activity of mischarged 3'-fluoro-A76 tRNAVal (Thr-3'F-A76) is so low that it is indistinguishable from the background rate. B, the modified tRNAs require higher concentrations of ValRS to attain similar deacylation activity as that of A76 tRNAVal.

To further pursue investigation of 3'-modified tRNAVal substrates, Thr-3'-fluoro-A76 tRNAVal was constructed. Little deacylation of Thr-3'-fluoro-A76 tRNAVal was detected over the relatively high rate of non-enzyme catalyzed deacylation (Fig. 6A). This result is consistent with the full mischarging of 3'-fluoro-A76 tRNAVal under conditions where 3'-dA76 tRNAVal is only partially mischarged. With higher concentrations of ValRS, Thr-3'-fluoro-A76 tRNAVal is indeed deacylated by ValRS (Fig 6B). Thus, ValRS has distinct substrate specificity relative to its close homolog IleRS. Whether ValRS simply has broader hydroxyl specificity or has reversed hydroxyl specificity relative to IleRS is unanswered. Attempts to mischarge 2'-dA76 tRNAVal by a variety of approaches and subsequently measure deacylation of Thr-2'-dA76 tRNAVal were unsuccessful.

The Editing Site of IleRS Can Adapt to Hydrolyze 2'-O-Acyl Linkages-- The results with ValRS raised the possibility that editing is inherently "plastic" and that, given the appropriate stimulus, IleRS might also deacylate substrates mischarged at the 2'-OH. Here, the minihelix system was of particular advantage relative to the use of tRNA substrates. A 3'-O-methyl-A76 minihelixIle (3'-OMe-A76) could be synthesized from commercially available starting materials, whereas 3'-OMe-ATP is not a substrate for tRNA NTase. A mischarged 3'-OMe-A76 minihelixIle has a methyl group that prevents a 2'- to 3'-transacylation from occurring. Although this molecule has an intact 2'-OH, it was not aminoacylated by IleRS. Successful aminoacylation was only achieved using yeast ValRS under the same conditions (30% Me2SO and no monovalent cations) that were used for mischarging 2'-dA76 substrates.

Interestingly, Val-3'-OMe-A76 minihelixIle was efficiently deacylated by IleRS (Fig. 7A). To verify that this deacylation activity was catalyzed by the editing site in CP1, we demonstrated that the editing-deficient mutant T242P IleRS was unable to deacylate Val-3'-OMe-A76 minihelixIle (data not shown). (The T242P mutation is located in the editing pocket of CP1 (12).) The rate of Val-3'-OMe-A76 minihelixIle deacylation is only 2-fold reduced from that of wild-type minihelixIle and, thus, significantly faster than the deacylation-resistant Val-3'-dA76 minihelixIle. Because the steric bulk of the O-methyl group could alter the positioning of the mischarged RNA terminus in the editing site so as to place the hydrolytic machinery in close proximity to the aminoacyl linkage, a search for conditions that might alter the position of the 2'-O-acyl linkage of Val-3'-dA76 tRNAIle within CP1 was undertaken. Importantly, Val-3'-dA76 tRNAIle is efficiently hydrolyzed in the presence of high enzyme concentrations and 20% methanol (Fig. 7B). However, it is worth noting that these conditions also promoted the hydrolysis of Ile-3'-dA76 tRNAIle. Thus, under these conditions the aminoacyl terminus is bound in a modified orientation.


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  		Fig. 7.   IleRS catalyzed deacylation of 2'-O-esters. A, IleRS can deacylate a mischarged 3'-OMe-A76 minihelixIle. The rate is ~2-fold reduced from that of the wild-type or 2'-dA76 minihelix. B, with 20% methanol added to the solvent, even a mischarged 3'-dA76 tRNAIle can be deacylated. Under these conditions the molecular recognition in the editing active site is disrupted to the point where properly charged 3'-dA76 tRNAIle is also deacylated.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A mischarged 3'-dA76 tRNAIle is completely resistant to deacylation by IleRS. This resistance is most likely because the aminoacyl linkage prevents the scissile ester bond from coming into close proximity of the hydrolytic machinery while bound in the editing site. The magnitude of the rate difference (>= 750-fold) between deacylation of Val-A76 tRNAIle and Val-3'-dA76 tRNAIle is larger than one might expect if the 3'-OH had a noncovalent role in deacylation. The calculated difference in transition state stabilization of ~4 kcal/mol is beyond the range of energies observed for average hydrogen bonds or the electronegative inductive effect of a neighboring hydroxyl. Correspondingly, a fluoro group in place of the 3'-OH failed to allow for even partial deacylation activity. Earlier work did not directly investigate the deacylation of Val-2'-dA76 tRNAIle and, given the available data, concluded that the role of the 3'-OH was to assist catalysis of deacylation from the 2'-OH (43). Here, reasonable quantities of mischarged 2'-dA76 tRNAIle were produced, isolated, and directly shown to be deacylated by IleRS. This mischarged substrate, with a fixed 3'-O-aminoacyl linkage, showed only a modest 5-fold decrease in initial rate of deacylation. This rate decrease is in the range of what might be expected for the loss of neighboring group effects due to the missing 2'-OH (20). Thus, under normal circumstances, transacylation from the 2'- to the 3'-OH is required for deacylation of Val-tRNAIle by IleRS.

Although IleRS cannot deacylate Val-3'-dA76 tRNAIle, ValRS deacylates Thr-3'-dA76 tRNAVal with an approximate 10-fold reduction in rate compared with deacylation of Thr-A76 tRNAVal. Still, it is possible that ValRS preferentially deacylates 3'-O-aminoacyl esters. Even though the initial site of aminoacylation is the 2'-OH, because transacylation is more rapid than deacylation (21), a 3'-O-aminoacyl ester could be the main substrate for editing. In that event, a 10-fold reduced deacylation rate of Thr-3'-dA76 tRNAVal relative to Thr-A76 tRNAVal would not be surprising.

Alternatively, the role of the 3'-OH of tRNAVal may be as a hydrogen bond donor (Fig. 8). In this model, derived from studies of non-catalyzed ester hydrolysis by Bruice and co-workers (20), the neighboring hydroxyl donates a hydrogen to the oxyanion of the tetrahedral intermediate, thereby helping to stabilize the build up of negative charge (Fig. 8, left). The 3'-dA analog is obviously unable to fulfill this role (Fig. 8, middle). Not only is the fluoro group unable to donate a hydrogen bond, but its partial negative charge likely also induces some electrostatic repulsion that distorts the tetrahedral intermediate (Fig. 8, right). This model fits best with the observed trends in deacylation rates for these substrates.


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  		Fig. 8.   Molecular interactions at the 3'-end of tRNAVal. The tetrahedral intermediate formed during hydrolysis of mischarged wild-type tRNAVal (left) is likely stabilized by intramolecular hydrogen bonding from the adjacent hydroxyl. During deacylation of mischarged 3'-dA76 tRNAVal (center) this hydrogen bonding is not possible and thus, the deacylation rate is decreased. The electronegativity of the fluorine atom in 3'-fluoro-A76 tRNAVal (right) would tend to have a repulsive effect on the anion of the tetrahedral intermediate. This likely explains the even further reduced rate of deacylation for this substrate.

The observed deacylation of Val-3'-OMe-A76 minihelixIle by IleRS demonstrates a shift in substrate specificity for deacylation reminiscent of the substrate specificity ValRS shows. The mechanism by which IleRS is able to hydrolyze this 2'-O-aminoacyl ester likely arises from some local structural rearrangement induced by the bulkier 3'-end as opposed to a hydrogen bonding role for the 3'-OMe group (the 3'-OH cannot play a similar hydrogen-bonding role during deacylation of Val-2'-dA76 substrates). In the context of a 2'-O-aminoacyl ester, the editing active site may be too crowded to accommodate the 3'-OMe group in the normal binding orientation. Alternatively, there could be a small hydrophobic interaction that is made with the 3'-O-Me group, also altering the position of the scissile ester bond with respect to the catalytic center. Such local structural perturbations are certainly feasible within the IleRS-editing site, as evidenced by the deacylation activity seen in the presence of 20% methanol.

Regardless of the detailed mechanism, the results show that the editing site is inherently plastic with respect to recognition of mischarged tRNA. For IleRS and ValRS to discriminate against valine and threonine, respectively, the editing sites of both enzymes must recognize subtle differences in aminoacyl side chains. Yet this fine structure discrimination must also be flexible, as both enzymes have been shown to edit multiple non-cognate amino acids (44). A third, related synthetase, leucyl-tRNA synthetase, has also been reported to edit multiple amino acids (45, 46). This remarkable combination of specificity and plasticity in recognition of the aminoacyl side chain is shown here to include the position of the aminoacyl group on the 3'-end of tRNA. This plasticity may have developed over a long period of evolution and may have been particularly important in the early stages of the development of aminoacylation systems and the genetic code, when aminoacyl-RNA substrates were not perfected. Indeed, the CP1 insertion is ancient, as it is conserved throughout evolution and found in deeply rooted organisms of all three kingdoms such as the Thermotogales, Crenarchaeota, and Diplomonads (47).

    ACKNOWLEDGEMENTS

We thank Dr. Nancy Maizels and Dr. Alan Weiner for providing the plasmid encoding tRNA NTase, and Dr. Chien-Chia Wang for cloning and expressing yeast ValRS. We are grateful to Dr. T. Hendrickson, Dr. T. Nomanbhoy, L. Nangle, and Dr. M. Sprinzl for help in preparing materials and insightful discussion.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM15539 and a fellowship from the National Foundation for Cancer Research.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.

Dagger Howard Hughes Medical Institute Predoctoral fellow.

§ To whom correspondence should be addressed. Tel.: 858-784-8970; Fax: 858-784-8990; E-mail: schimmel@scripps.edu.

Published, JBC Papers in Press, March 28, 2002, DOI 10.1074/jbc.M202023200

2 C.-C. Wang, unpublished data.

    ABBREVIATIONS

The abbreviations used are: IleRS, isoleucyl-tRNA synthetase; ValRS, valyl-tRNA synthetase; CP1, connective polypeptide 1; tRNA NTase, tRNA nucleotidyltransferase; 2'-dA, 2'-deoxyadenosine; 3'-dA 3'-deoxyadenosine, 2'-fluoro-A, 2'-deoxy-2'-fluoroadenosine; 3'-fluoro-A, 3'-deoxy-3'-fluoroadenosine; 3'-OMe-A, 3'-deoxy-3'-O-methyl-adenosine.

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RESULTS
DISCUSSION
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