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• W2034361496 abstract "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. 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. The genetic code is based on the accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetases (1Carter C.W., Jr. Annu. Rev. Biochem. 1993; 62: 715-748Crossref PubMed Scopus (327) Google Scholar, 2Giegé R. Puglisi J.D. Florentz C. Prog. Nucleic Acids Res. Mol. Biol. 1993; 45: 129-206Crossref PubMed Scopus (218) Google Scholar). 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 (3Pauling L. Festschrift fuer Prof. Dr. Arthur Stoll. Birkhauser Verlag, Basel, Switzerland1958Google Scholar) 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 coliIleRS 1The abbreviations used are: IleRSisoleucyl-tRNA synthetaseValRSvalyl-tRNA synthetaseCP1connective polypeptide 1tRNA NTasetRNA nucleotidyltransferase2′-dA2′-deoxyadenosine3′-dA 3′-deoxyadenosine2′-fluoro-A, 2′-deoxy-2′-fluoroadenosine3′-fluoro-A3′-deoxy-3′-fluoroadenosine3′-OMe-A3′-deoxy-3′-O-methyl-adenosine1The abbreviations used are: IleRSisoleucyl-tRNA synthetaseValRSvalyl-tRNA synthetaseCP1connective polypeptide 1tRNA NTasetRNA nucleotidyltransferase2′-dA2′-deoxyadenosine3′-dA 3′-deoxyadenosine2′-fluoro-A, 2′-deoxy-2′-fluoroadenosine3′-fluoro-A3′-deoxy-3′-fluoroadenosine3′-OMe-A3′-deoxy-3′-O-methyl-adenosine only 180-fold less efficiently than isoleucine (4Schmidt E. Schimmel P. Science. 1994; 264: 265-267Crossref PubMed Scopus (138) Google Scholar). However, the substitution of valine for isoleucine at isoleucine codons in the cell is less than 1 in 3000 (5Loftfield R.B. Biochem. J. 1963; 89: 82-92Crossref PubMed Scopus (116) Google Scholar). The increased specificity is a result of the RNA-dependent editing of misactivated valine by IleRS (6Baldwin A.N. Berg P. J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar,7Eldred E.W. Schimmel P.R. J. Biol. Chem. 1972; 247: 2961-2964Abstract Full Text PDF PubMed Google Scholar). 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 (8Fersht A.R. Dingwall C. Biochemistry. 1979; 18: 2627-2631Crossref PubMed Scopus (120) Google Scholar). Like IleRS, ValRS prevents the misincorporation of threonine into proteins through the RNA-dependent editing of misactivated threonine (9Fersht A.R. Kaethner M.M. Biochemistry. 1976; 15: 3342-3346Crossref PubMed Scopus (180) Google Scholar).These reactions strictly require the presence of the cognate tRNA (6Baldwin A.N. Berg P. J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar,10Hale S.P. Auld D.S. Schmidt E. Schimmel P. Science. 1997; 276: 1250-1252Crossref PubMed Scopus (79) Google Scholar). 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 (7Eldred E.W. Schimmel P.R. J. Biol. Chem. 1972; 247: 2961-2964Abstract Full Text PDF PubMed Google Scholar, 9Fersht A.R. Kaethner M.M. Biochemistry. 1976; 15: 3342-3346Crossref PubMed Scopus (180) Google Scholar). 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 (11Lin L. Hale S.P. Schimmel P. Nature. 1996; 384: 33-34Crossref PubMed Scopus (92) Google Scholar). 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 (12Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (314) Google Scholar). This site binds valine but sterically excludes isoleucine. A co-crystal structure of Staphylococcus aureus IleRS with tRNAIlesuggested how the tRNA may place its 3′-end in the editing site (13Silvian L.F. Wang J. Steitz T.A. Science. 1999; 285: 1074-1077Crossref PubMed Scopus (351) Google Scholar). 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 (14Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Although a mischarged amino acid could be modeled at the end of the tRNA, neither this model nor mutational analysis established a hydrolytic mechanism (15Schmidt E. Schimmel P. Biochemistry. 1995; 34: 11204-11210Crossref PubMed Scopus (78) Google Scholar,16Hendrickson T.L. Nomanbhoy T.K. Schimmel P. Biochemistry. 2000; 39: 8180-8186Crossref PubMed Scopus (45) Google Scholar).Early work demonstrated an important role for the 3′-end of tRNAIle and tRNAVal in editing (17von der Haar F. Cramer F. FEBS Lett. 1975; 56: 215-217Crossref PubMed Scopus (33) Google Scholar, 18von der Haar F. Cramer F. Biochemistry. 1976; 15: 4131-4138Crossref PubMed Scopus (80) Google Scholar, 19Igloi G.L. von der Haar F. Cramer F. Biochemistry. 1977; 16: 1696-1702Crossref PubMed Scopus (42) Google Scholar). 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 (20Bruice T.C. Fife T.H. J. Am. Chem. Soc. 1962; 84: 1973-1979Crossref Scopus (86) Google Scholar). 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) (21Taiji M. Yokoyama S. Miyazawa T. Biochemistry. 1983; 22: 3220-3225Crossref PubMed Scopus (58) Google Scholar). Thus, while aminoacylation is specific to a particular hydroxyl (2′-OH in the cases of IleRS and ValRS (22Cramer F. Faulhammer H. von der Haar F. Sprinzl M. Sternbach H. FEBS Lett. 1975; 56: 212-214Crossref PubMed Scopus (41) Google Scholar)) 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.DISCUSSIONA 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 (43Freist W. Cramer F. Eur. J. Biochem. 1983; 131: 65-80Crossref PubMed Scopus (17) Google Scholar). 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 (20Bruice T.C. Fife T.H. J. Am. Chem. Soc. 1962; 84: 1973-1979Crossref Scopus (86) Google Scholar). 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 (21Taiji M. Yokoyama S. Miyazawa T. Biochemistry. 1983; 22: 3220-3225Crossref PubMed Scopus (58) Google Scholar), 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 (20Bruice T.C. Fife T.H. J. Am. Chem. Soc. 1962; 84: 1973-1979Crossref Scopus (86) Google Scholar), 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.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 (44Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar). A third, related synthetase, leucyl-tRNA synthetase, has also been reported to edit multiple amino acids (45Englisch S. Englisch U. von der Haar F. Cramer F. Nucleic Acids Res. 1986; 14: 7529-7539Crossref PubMed Scopus (78) Google Scholar, 46Mursinna R.S. Lincecum T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar). 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 (47Schimmel, P., and Ribas de Pouplanas, L. (2002) Cold Spring Harbor Symp. Quant. Biol., in pressGoogle Scholar). The genetic code is based on the accurate aminoacylation of tRNAs by aminoacyl-tRNA synthetases (1Carter C.W., Jr. Annu. Rev. Biochem. 1993; 62: 715-748Crossref PubMed Scopus (327) Google Scholar, 2Giegé R. Puglisi J.D. Florentz C. Prog. Nucleic Acids Res. Mol. Biol. 1993; 45: 129-206Crossref PubMed Scopus (218) Google Scholar). 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 (3Pauling L. Festschrift fuer Prof. Dr. Arthur Stoll. Birkhauser Verlag, Basel, Switzerland1958Google Scholar) 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 coliIleRS 1The abbreviations used are: IleRSisoleucyl-tRNA synthetaseValRSvalyl-tRNA synthetaseCP1connective polypeptide 1tRNA NTasetRNA nucleotidyltransferase2′-dA2′-deoxyadenosine3′-dA 3′-deoxyadenosine2′-fluoro-A, 2′-deoxy-2′-fluoroadenosine3′-fluoro-A3′-deoxy-3′-fluoroadenosine3′-OMe-A3′-deoxy-3′-O-methyl-adenosine1The abbreviations used are: IleRSisoleucyl-tRNA synthetaseValRSvalyl-tRNA synthetaseCP1connective polypeptide 1tRNA NTasetRNA nucleotidyltransferase2′-dA2′-deoxyadenosine3′-dA 3′-deoxyadenosine2′-fluoro-A, 2′-deoxy-2′-fluoroadenosine3′-fluoro-A3′-deoxy-3′-fluoroadenosine3′-OMe-A3′-deoxy-3′-O-methyl-adenosine only 180-fold less efficiently than isoleucine (4Schmidt E. Schimmel P. Science. 1994; 264: 265-267Crossref PubMed Scopus (138) Google Scholar). However, the substitution of valine for isoleucine at isoleucine codons in the cell is less than 1 in 3000 (5Loftfield R.B. Biochem. J. 1963; 89: 82-92Crossref PubMed Scopus (116) Google Scholar). The increased specificity is a result of the RNA-dependent editing of misactivated valine by IleRS (6Baldwin A.N. Berg P. J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar,7Eldred E.W. Schimmel P.R. J. Biol. Chem. 1972; 247: 2961-2964Abstract Full Text PDF PubMed Google Scholar). 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 (8Fersht A.R. Dingwall C. Biochemistry. 1979; 18: 2627-2631Crossref PubMed Scopus (120) Google Scholar). Like IleRS, ValRS prevents the misincorporation of threonine into proteins through the RNA-dependent editing of misactivated threonine (9Fersht A.R. Kaethner M.M. Biochemistry. 1976; 15: 3342-3346Crossref PubMed Scopus (180) Google Scholar). isoleucyl-tRNA synthetase valyl-tRNA synthetase connective polypeptide 1 tRNA nucleotidyltransferase 2′-deoxyadenosine 2′-fluoro-A, 2′-deoxy-2′-fluoroadenosine 3′-deoxy-3′-fluoroadenosine 3′-deoxy-3′-O-methyl-adenosine isoleucyl-tRNA synthetase valyl-tRNA synthetase connective polypeptide 1 tRNA nucleotidyltransferase 2′-deoxyadenosine 2′-fluoro-A, 2′-deoxy-2′-fluoroadenosine 3′-deoxy-3′-fluoroadenosine 3′-deoxy-3′-O-methyl-adenosine These reactions strictly require the presence of the cognate tRNA (6Baldwin A.N. Berg P. J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar,10Hale S.P. Auld D.S. Schmidt E. Schimmel P. Science. 1997; 276: 1250-1252Crossref PubMed Scopus (79) Google Scholar). 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 (7Eldred E.W. Schimmel P.R. J. Biol. Chem. 1972; 247: 2961-2964Abstract Full Text PDF PubMed Google Scholar, 9Fersht A.R. Kaethner M.M. Biochemistry. 1976; 15: 3342-3346Crossref PubMed Scopus (180) Google Scholar). 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 (11Lin L. Hale S.P. Schimmel P. Nature. 1996; 384: 33-34Crossref PubMed Scopus (92) Google Scholar). 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 (12Nureki O. Vassylyev D.G. Tateno M. Shimada A. Nakama T. Fukai S. Konno M. Hendrickson T.L. Schimmel P. Yokoyama S. Science. 1998; 280: 578-582Crossref PubMed Scopus (314) Google Scholar). This site binds valine but sterically excludes isoleucine. A co-crystal structure of Staphylococcus aureus IleRS with tRNAIlesuggested how the tRNA may place its 3′-end in the editing site (13Silvian L.F. Wang J. Steitz T.A. Science. 1999; 285: 1074-1077Crossref PubMed Scopus (351) Google Scholar). 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 (14Fukai S. Nureki O. Sekine S. Shimada A. Tao J. Vassylyev D.G. Yokoyama S. Cell. 2000; 103: 793-803Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Although a mischarged amino acid could be modeled at the end of the tRNA, neither this model nor mutational analysis established a hydrolytic mechanism (15Schmidt E. Schimmel P. Biochemistry. 1995; 34: 11204-11210Crossref PubMed Scopus (78) Google Scholar,16Hendrickson T.L. Nomanbhoy T.K. Schimmel P. Biochemistry. 2000; 39: 8180-8186Crossref PubMed Scopus (45) Google Scholar). Early work demonstrated an important role for the 3′-end of tRNAIle and tRNAVal in editing (17von der Haar F. Cramer F. FEBS Lett. 1975; 56: 215-217Crossref PubMed Scopus (33) Google Scholar, 18von der Haar F. Cramer F. Biochemistry. 1976; 15: 4131-4138Crossref PubMed Scopus (80) Google Scholar, 19Igloi G.L. von der Haar F. Cramer F. Biochemistry. 1977; 16: 1696-1702Crossref PubMed Scopus (42) Google Scholar). 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 (20Bruice T.C. Fife T.H. J. Am. Chem. Soc. 1962; 84: 1973-1979Crossref Scopus (86) Google Scholar). 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) (21Taiji M. Yokoyama S. Miyazawa T. Biochemistry. 1983; 22: 3220-3225Crossref PubMed Scopus (58) Google Scholar). Thus, while aminoacylation is specific to a particular hydroxyl (2′-OH in the cases of IleRS and ValRS (22Cramer F. Faulhammer H. von der Haar F. Sprinzl M. Sternbach H. FEBS Lett. 1975; 56: 212-214Crossref PubMed Scopus (41) Google Scholar)) 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. DISCUSSIONA 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 (43Freist W. Cramer F. Eur. J. Biochem. 1983; 131: 65-80Crossref PubMed Scopus (17) Google Scholar). 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 (20Bruice T.C. Fife T.H. J. Am. Chem. Soc. 1962; 84: 1973-1979Crossref Scopus (86) Google Scholar). 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 (21Taiji M. Yokoyama S. Miyazawa T. Biochemistry. 1983; 22: 3220-3225Crossref PubMed Scopus (58) Google Scholar), 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 (20Bruice T.C. Fife T.H. J. Am. Chem. Soc. 1962; 84: 1973-1979Crossref Scopus (86) Google Scholar), 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.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 (44Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar). A third, related synthetase, leucyl-tRNA synthetase, has also been reported to edit multiple amino acids (45Englisch S. Englisch U. von der Haar F. Cramer F. Nucleic Acids Res. 1986; 14: 7529-7539Crossref PubMed Scopus (78) Google Scholar, 46Mursinna R.S. Lincecum T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar). 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 (47Schimmel, P., and Ribas de Pouplanas, L. (2002) Cold Spring Harbor Symp. Quant. Biol., in pressGoogle Scholar). 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 (43Freist W. Cramer F. Eur. J. Biochem. 1983; 131: 65-80Crossref PubMed Scopus (17) Google Scholar). 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 (20Bruice T.C. Fife T.H. J. Am. Chem. Soc. 1962; 84: 1973-1979Crossref Scopus (86) Google Scholar). 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 (21Taiji M. Yokoyama S. Miyazawa T. Biochemistry. 1983; 22: 3220-3225Crossref PubMed Scopus (58) Google Scholar), 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 (20Bruice T.C. Fife T.H. J. Am. Chem. Soc. 1962; 84: 1973-1979Crossref Scopus (86) Google Scholar), 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. 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 (44Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar). A third, related synthetase, leucyl-tRNA synthetase, has also been reported to edit multiple amino acids (45Englisch S. Englisch U. von der Haar F. Cramer F. Nucleic Acids Res. 1986; 14: 7529-7539Crossref PubMed Scopus (78) Google Scholar, 46Mursinna R.S. Lincecum T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar). 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 (47Schimmel, P., and Ribas de Pouplanas, L. (2002) Cold Spring Harbor Symp. Quant. Biol., in pressGoogle Scholar). 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." @default.
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• W2034361496 title "Plasticity of Recognition of the 3′-End of Mischarged tRNA by Class I Aminoacyl-tRNA Synthetases" @default.
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