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RNA Determinants for Translational Editing

1999; Elsevier BV; Volume: 274; Issue: 11 Linguagem: Inglês

10.1074/jbc.274.11.6835

1083-351X

Brian E. Nordin, Paul Schimmel,

RNA modifications and cancer

The fidelity of protein synthesis requires efficient discrimination of amino acid substrates by aminoacyl-tRNA synthetases. Accurate discrimination of the structurally similar amino acids, valine and isoleucine, by isoleucyl-tRNA synthetase (IleRS) results, in part, from a hydrolytic editing reaction, which prevents misactivated valine from being stably joined to tRNAIle. The editing reaction is dependent on the presence of tRNAIle, which contains discrete D-loop nucleotides that are necessary to promote editing of misactivated valine. RNA minihelices comprised of just the acceptor-TΨC helix of tRNAIle are substrates for specific aminoacylation by IleRS. These substrates lack the aforementioned D-loop nucleotides. Because minihelices contain determinants for aminoacylation, we thought that they might also play a role in editing that has not previously been recognized. Here we show that, in contrast to tRNAIle, minihelixIle is unable to trigger the hydrolysis of misactivated valine and, in fact, is mischarged with valine. In addition, mutations in minihelixIle that enhance or suppress charging with isoleucine do the same with valine. Thus, minihelixIle contains signals for charging (by IleRS) that are independent of the amino acid and, by itself, minihelixIle provides no determinants for editing. An RNA hairpin that mimics the D-stem/loop of tRNAIle is also unable to induce the hydrolysis of misactivated valine, both by itself and in combination with minihelixIle. Thus, the native tertiary fold of tRNAIle is required to promote efficient editing. Considering that the minihelix is thought to be the more ancestral part of the tRNA structure, these results are consistent with the idea that, during the development of the genetic code, RNA determinants for editing were added after the establishment of an aminoacylation system. The fidelity of protein synthesis requires efficient discrimination of amino acid substrates by aminoacyl-tRNA synthetases. Accurate discrimination of the structurally similar amino acids, valine and isoleucine, by isoleucyl-tRNA synthetase (IleRS) results, in part, from a hydrolytic editing reaction, which prevents misactivated valine from being stably joined to tRNAIle. The editing reaction is dependent on the presence of tRNAIle, which contains discrete D-loop nucleotides that are necessary to promote editing of misactivated valine. RNA minihelices comprised of just the acceptor-TΨC helix of tRNAIle are substrates for specific aminoacylation by IleRS. These substrates lack the aforementioned D-loop nucleotides. Because minihelices contain determinants for aminoacylation, we thought that they might also play a role in editing that has not previously been recognized. Here we show that, in contrast to tRNAIle, minihelixIle is unable to trigger the hydrolysis of misactivated valine and, in fact, is mischarged with valine. In addition, mutations in minihelixIle that enhance or suppress charging with isoleucine do the same with valine. Thus, minihelixIle contains signals for charging (by IleRS) that are independent of the amino acid and, by itself, minihelixIle provides no determinants for editing. An RNA hairpin that mimics the D-stem/loop of tRNAIle is also unable to induce the hydrolysis of misactivated valine, both by itself and in combination with minihelixIle. Thus, the native tertiary fold of tRNAIle is required to promote efficient editing. Considering that the minihelix is thought to be the more ancestral part of the tRNA structure, these results are consistent with the idea that, during the development of the genetic code, RNA determinants for editing were added after the establishment of an aminoacylation system. Aminoacyl-tRNA synthetases establish the genetic code by attaching amino acids to their cognate tRNAs (1Lapointe J. Giegé R. Trachsel H. Translation in Eukaryotes. CRC Press, Inc., Boca Raton, FL1991: 35-69Google Scholar, 2Giegé R. Puglisi J.D. Florentz C. Prog. Nucleic Acid Res. Mol. Biol. 1993; 45: 129-206Crossref PubMed Scopus (218) Google Scholar, 3Carter Jr., C.W. Annu. Rev. Biochem. 1993; 62: 715-748Crossref PubMed Scopus (328) Google Scholar). These reactions are comprised of two steps. Initially the amino acid is condensed with ATP to give an activated aminoacyl adenylate. Subsequently, the aminoacyl moiety of this reactive intermediate is transesterified to the 3′ terminus of the tRNA. The fidelity of the genetic code depends upon the precise molecular recognition of both the amino acid and tRNA substrates by aminoacyl-tRNA synthetases. It has long been recognized that the accurate transduction of molecular information is made increasingly difficult when the molecular structures of two candidate substrates are highly similar (4Pauling L. Festschrift fuer Prof. Dr. Arthur Stoll. Birkhauser Verlag, Basel1958: 597-602Google Scholar, 5Cramer F. Englisch U. Freist W. Sternbach H. Biochimie (Paris). 1991; 73: 1027-1035Crossref PubMed Scopus (18) Google Scholar, 6Jakubowski H. Fersht A.R. Nucleic Acids Res. 1981; 9: 3105-3117Crossref PubMed Scopus (136) Google Scholar, 7Fersht A. Enzyme Structure and Mechanism. 2nd Ed. W. H. Freeman and Co., New York1985: 355-358Google Scholar). A prominent example of the need to discriminate between closely related substrates is in the recognition of isoleucine over valine by IleRS. 1The abbreviations used are: IleRS, isoleucyl-tRNA synthetase; Ile-AMP, isoleucyl adenylate; Val-AMP, valyl adenylate Valine, which differs from isoleucine by a single methylene unit, is activated byEscherichia coli IleRS at a rate approximately 180-fold slower than that of isoleucine (8Schmidt E. Schimmel P. Science. 1994; 264: 265-267Crossref PubMed Scopus (138) Google Scholar). IleRS prevents this relatively high error rate from being realized in protein synthesis through two editing reactions that result in the net hydrolysis of misactivated valine (7Fersht A. Enzyme Structure and Mechanism. 2nd Ed. W. H. Freeman and Co., New York1985: 355-358Google Scholar,9Baldwin A.N. Berg P. J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar, 10Eldred E.W. Schimmel P.R. J. Biol. Chem. 1972; 247: 2961-2964Abstract Full Text PDF PubMed Google Scholar). These reactions provide a second "sieve" (11Fersht A.R. Dingwall C. Biochemistry. 1979; 18: 2627-2631Crossref PubMed Scopus (120) Google Scholar) of discrimination against valine and indeed occur at a second, "editing" active site on IleRS (8Schmidt E. Schimmel P. Science. 1994; 264: 265-267Crossref PubMed Scopus (138) Google Scholar, 12Schmidt E. Schimmel P. Biochemistry. 1995; 34: 11204-11210Crossref PubMed Scopus (78) Google Scholar). Editing of misactivated valine has a strict requirement for tRNAIle (9Baldwin A.N. Berg P. J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar, 13Hale S.P. Auld D.S. Schmidt E. Schimmel P. Science. 1997; 276: 1250-1252Crossref PubMed Scopus (79) Google Scholar). In the absence of tRNAIle, enzymatically generated Ile-AMP and Val-AMP remain sequestered in the active site. Upon addition of tRNAIle to the IleRS·Ile-AMP complex, the aminoacyl group is stably attached to tRNAIle. In contrast, addition of tRNAIle to the IleRS·Val-AMP complex results in immediate hydrolysis of the valyl adenylate. This occurs partly through a pretransfer editing reaction where misactivated Val-AMP is directly hydrolyzed. Alternatively, the valyl moiety of Val-AMP is transferred to tRNAIle to make a transient Val-tRNAIleintermediate that is rapidly hydrolyzed. The net result of either pathway is an abortive cycle of valine activation followed by tRNAIle-dependent hydrolysis that continues until all available ATP is consumed. In contrast to nucleotide determinants for charging that are located in the anticodon loop and acceptor stem of tRNAIle (14Nureki O. Niimi T. Muramatsu T. Kanno H. Kohno T. Florentz C. Giegé R. Yokoyama S. J. Mol. Biol. 1994; 236: 710-724Crossref PubMed Scopus (101) Google Scholar), nucleotides in the D-loop of tRNAIle trigger the editing reaction (13Hale S.P. Auld D.S. Schmidt E. Schimmel P. Science. 1997; 276: 1250-1252Crossref PubMed Scopus (79) Google Scholar). For example, replacement of G-16, D-20, and D-21 in the D-loop of tRNAIle with their counterparts from tRNAVal has little effect on aminoacylation. In contrast, these substitutions abolish the editing response. Conversely, transfer of the three D-loop nucleotides from tRNAIle into the framework of a specially designed tRNAVal confers editing activity to the chimerized tRNA. Thus, tRNA determinants for editing and aminoacylation are discrete. Similarly, a single (G56A) mutation in the active site (for aminoacylation) of IleRS eliminates the discrimination between isoleucine and valine in amino acid activation (8Schmidt E. Schimmel P. Science. 1994; 264: 265-267Crossref PubMed Scopus (138) Google Scholar). However, the G56A mutant enzyme still discriminates between isoleucine and valine in the post-transfer editing reaction. These and other mutational analyses, along with chemical cross-linking data (12Schmidt E. Schimmel P. Biochemistry. 1995; 34: 11204-11210Crossref PubMed Scopus (78) Google Scholar, 15Hale S.P. Schimmel P. Tetrahedron. 1997; 53: 11985-11994Crossref Scopus (13) Google Scholar), showed that the editing and aminoacylation sites are physically distinct and functionally independent. Specifically, the editing site is located within a large insertion known as CP1 (connective polypeptide 1) (16Lin L. Hale S.P. Schimmel P. Nature. 1996; 384: 33-34Crossref PubMed Scopus (92) Google Scholar). This 277-amino acid insertion divides the characteristic class I catalytic domain in half (17Starzyk R.M. Webster T.A. Schimmel P. Science. 1987; 237: 1614-1618Crossref PubMed Scopus (141) Google Scholar). A recently determined x-ray structure places the two active sites about 25-Å apart (18Nureki 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 (315) Google Scholar). A present working hypothesis is that specific nucleotides in the D-loop of tRNAIle trigger the translocation of the valyl group from the aminoacylation to the editing site. Here we tested whether the presumptive translocation and editing response could be recreated by dividing the critical domains of tRNAIle into two pieces. One piece is an oligonucleotide substrate that recreates the acceptor stem of the tRNA in the form of a minihelix. Previous work showed that IleRS could charge minihelixIle with isoleucine (19Nureki O. Niimi T. Muto Y. Kanno H. Kohno T. Muramatsu T. Kawai G. Miyazawa T. Giegé R. Florentz C. Yokoyama S. Nierhaus K.H. Franceschi F. Subramanian A.R. Erdmann V.A. Wittman-Liebold B. The Translational Apparatus. Plenum Publishing Corp., New York1993: 59-66Crossref Google Scholar, 20Alexander R.W. Nordin B.E. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12214-12219Crossref PubMed Scopus (21) Google Scholar). The other piece is an RNA hairpin ligand designed after the D-loop of tRNAIle. Thus, we asked whether the two pieces in concert could reproduce the editing reaction or whether continuity of the tRNA structure was required. If the editing response requires the full tRNAIle structure and if the D-loop provides critical determinants only within the context of the full tRNA, then we imagined that minihelixIle might be a good substrate for mischarging with valine. In that event, we wondered whether mischarging depended on the same nucleotide determinants as those required for correct aminoacylation. This part of the analysis was motivated by the prospect of discovering acceptor stem determinants that modulate discrimination against valine. Wild-type E. coli IleRS was overexpressed in the E. coli strain MV1184 harboring the multicopy plasmid pKS21 (21Shiba K. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1880-1884Crossref PubMed Scopus (110) Google Scholar). Protein purification was essentially as described previously (22Shepard A. Shiba K. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9964-9968Crossref PubMed Scopus (45) Google Scholar). RNA hairpins were chemically synthesized using N-phenoxyacetyl-protected ribonucleoside phosphoramidites (ChemGenes, Waltham, MA) on a Amersham Pharmacia Biotech Gene Assembler Special (23Wincott F. DiRenzo A. Shaffer C. Grimm S. Tracz D. Workman C. Sweedler D. Gonzalez C. Scaringe S. Usman N. Nucleic Acids Res. 1995; 23: 2677-2684Crossref PubMed Scopus (432) Google Scholar). RNA concentrations were determined using absorbance at 260 nm at room temperature. Extinction coefficients were estimated using the Biopolymer Calculator available online. 2A. Schepartz, web page address:http://paris.chem.yale.edu/extinct.html. Mature E. coli tRNA1Ile (GAU) was isolated from E. coli strain MV1184 containing the plasmid pES300, which allows for the lac-inducible overexpression of tRNAIle (24.Schmidt, E., Amino Acid Recognition by a Class I tRNA Synthetase. Ph.D. thesis, 1996, 160, Massachusetts Institute of Technology, Cambridge, MA.Google Scholar, 25Glasfeld E. Landro J.A. Schimmel P. Biochemistry. 1996; 35: 4139-4145Crossref PubMed Scopus (22) Google Scholar). The RNA-dependent hydrolysis of valyl adenylate was assayed by following the consumption of [γ-32P]ATP using a protocol that has been described in detail elsewhere (26Hale S.P. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2755-2758Crossref PubMed Scopus (43) Google Scholar). The reaction mixture contained 140 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 0.5 mm CaCl2, 2 mm[γ-32P]ATP (25 μCi/μmol), 1 mm valine, 75 nm inorganic pyrophosphatase, 5 μm IleRS, and either 40 μm tRNAIle or 200 μm minihelixIle and/or D-loopIle. Reactions with no RNA were used to control for a small background rate of hydrolysis (typically about 1% of the tRNAIle-dependent rate). The IleRS-catalyzed isoleucylation or valylation of RNA substrates was followed using the trichloroacetic acid precipitation method of Shepard and co-workers (22Shepard A. Shiba K. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9964-9968Crossref PubMed Scopus (45) Google Scholar). Reactions were carried out at room temperature in a solution containing 20 mm HEPES (pH 7.5), 150 mm NH4Cl, 20 mm MgCl2, 100 μm EDTA, 2 mm ATP, 10 nm inorganic pyrophosphatase, 5 μm [3H]amino acid (∼20 mCi/μmol), and 5 μm IleRS. RNA minihelices were used at a concentration of 50–500 μm and tRNAIle at a concentration of 10–40 μm. No-RNA controls were used to correct for background rates. The L-shaped tRNAIle structure (Fig. 1, top right) is composed of two helical domains (27Kim S.H. Suddath F.L. Quigley G.J. McPherson A. Sussman J.L. Wang A.H. Seeman N.C. Rich A. Science. 1974; 185: 435-440Crossref PubMed Scopus (754) Google Scholar, 28Robertus J.D. Ladner J.E. Finch J.T. Rhodes D. Brown R.S. Clark B.F. Klug A. Nature. 1974; 250: 546-551Crossref PubMed Scopus (803) Google Scholar, 29Rich A. RajBhandary U.L. Annu. Rev. Biochem. 1976; 45: 805-860Crossref PubMed Scopus (462) Google Scholar). Coaxial stacking of the acceptor and TΨC stems gives rise to the hairpin minihelix domain as the upper arm of the L-shape. The second domain is a "dumbbell" formed by the anticodon and D-stem/loops. Specific hydrogen bonds between the TΨC and D-loops link the two helical domains at the corner of the L-shaped tRNA. Interactions at the corner are crucial for establishing the canonical tRNA fold, and thus the nucleotides that form these interactions are well conserved among all tRNA species. This junction forms an obvious connection through which signals might be transmitted from one domain to the other. D-loop nucleotides previously identified as essential for editing are marked with arrows in Fig. 1. These nucleotides are not among those needed for the universal connections between the two domains. At least half of the tRNA synthetases charge minihelices based on the acceptor-TΨC stem of their cognate tRNAs (30Martinis S.A. Schimmel P. Söll D. RajBhandary U.L. tRNA: Structure, Biosynthesis, and Function. American Society for Microbiology, Washington, D. C.1995: 349-370Google Scholar, 31Frugier M. Florentz C. Giegé R. EMBO J. 1994; 13: 2218-2226Crossref PubMed Scopus (72) Google Scholar, 32Hamann C.S. Hou Y.M. Biochemistry. 1995; 34: 6527-6532Crossref PubMed Scopus (56) Google Scholar, 33Saks M.E. Sampson J.R. EMBO J. 1996; 15: 2843-2849Crossref PubMed Scopus (68) Google Scholar). Although certain synthetases do not make any contacts with the anticodon (e.g. alanyl-tRNA synthetase (34Park S.J. Schimmel P. J. Biol. Chem. 1988; 263: 16527-16530Abstract Full Text PDF PubMed Google Scholar) and seryl-tRNA synthetase (35Biou V. Yaremchuk A. Tukalo M. Cusack S. Science. 1994; 263: 1404-1410Crossref PubMed Scopus (425) Google Scholar)), many others interact with both the acceptor stem and anticodon. In the latter cases, the enzymes can still aminoacylate their associated minihelices, although the efficiency is generally severely reduced relative to the full tRNA. (In the E. coliisoleucine system studied here, the minihelix is ∼106-fold less active than tRNAIle.) Nevertheless, aminoacylation of minihelices generally retains the same sequence specificity for acceptor stem nucleotides as seen in the charging of tRNAs. For this reason, minihelices are thought to interact with the active site in a way that closely parallels the full tRNA. MinihelixIle (Fig. 1, bottom) contains determinants for aminoacylation by IleRS (19Nureki O. Niimi T. Muto Y. Kanno H. Kohno T. Muramatsu T. Kawai G. Miyazawa T. Giegé R. Florentz C. Yokoyama S. Nierhaus K.H. Franceschi F. Subramanian A.R. Erdmann V.A. Wittman-Liebold B. The Translational Apparatus. Plenum Publishing Corp., New York1993: 59-66Crossref Google Scholar, 20Alexander R.W. Nordin B.E. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12214-12219Crossref PubMed Scopus (21) Google Scholar). Previously, this domain of tRNAIle had not been investigated for its ability to stimulate editing of misactivated valine. As can be seen in Fig. 2, the addition of tRNAIle to a mixture of IleRS, ATP, and valine rapidly leads to the complete consumption of available ATP. In contrast, minihelixIle is unable to stimulate the hydrolytic editing of misactivated valine, even at the high concentration of 200 μm. Next we tested whether the addition of an isolated D-stem/loop domain of tRNAIle could induce editing. For this purpose, we constructed a 9-base pair RNA hairpin that extends the D-stem by pairing nucleotide G-26 and including 4 base pairs of the anticodon stem (D-loopIle, Fig. 1, top left). This D-stem/loop RNA hairpin did not induce editing (see Fig. 2). Finally, we obtained no evidence for an editing response when minihelixIle and D-loopIle were used in combination (data not shown for clarity). These results suggest that continuity of the tRNAIle structure is required for the editing response. Because minihelixIle is capable of being aminoacylated with isoleucine and yet is unable to induce editing, we tested its ability to be aminoacylated with valine. As shown in Fig. 3, minihelixIle is a relatively robust substrate for mischarging with valine. This level of mischarging was not observed with mature tRNAIle, as the editing reaction prevents the stable attachment of valine to tRNAIle. We tried to force misacylation of tRNAIle by using concentrations (40 μm) well above the K m (∼5 μm, Ref. 14Nureki O. Niimi T. Muramatsu T. Kanno H. Kohno T. Florentz C. Giegé R. Yokoyama S. J. Mol. Biol. 1994; 236: 710-724Crossref PubMed Scopus (101) Google Scholar). Still, no misacylation of the full tRNA could be detected. Thus, although minihelixIle is significantly less active than tRNAIle for charging with isoleucine, it is far more active in mischarging with valine. Despite the reduced activity of minihelixIle for charging with isoleucine, this aminoacylation is specific. For example, substitution of the A-73 discriminator with G results in a minihelix that is 8.4-fold less active in charging with isoleucine. A qualitatively similar effect is seen when the same substitution is made in the full tRNA (14Nureki O. Niimi T. Muramatsu T. Kanno H. Kohno T. Florentz C. Giegé R. Yokoyama S. J. Mol. Biol. 1994; 236: 710-724Crossref PubMed Scopus (101) Google Scholar). Here we found that mischarging A73G minihelixIlewith valine showed the same rate reduction that was observed for charging with isoleucine (Fig. 4). In addition, we investigated a variant of minihelixIledesignated Δ1 minihelixIle, in which the 5′-terminal A-1 nucleotide has been deleted. Recently, we discovered that this deletion enhances charging with isoleucine (20Alexander R.W. Nordin B.E. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12214-12219Crossref PubMed Scopus (21) Google Scholar). Presumably, this disruption of the first base pair increases the flexibility of the single-stranded 3′ terminus. (The 3′-end of bound tRNAGln in the co-crystal with glutaminyl-tRNA synthetase is folded back into the active site (36Rould M.A. Perona J.J. Söll D. Steitz T.A. Science. 1989; 246: 1135-1142Crossref PubMed Scopus (804) Google Scholar). Because glutaminyl-tRNA synthetase and IleRS are structurally related, the enhanced flexibility of the Δ1 substrate is thought to ease passage of the minihelix acceptor terminus into the transition state for catalysis.) The enhancement observed in charging Δ1 minihelixIle with isoleucine was exactly paralleled in misacylation with valine (Fig. 4). Thus, the determinants for charging of minihelixIle are independent of which amino acid is used as the substrate. These results rule out the possibility that the acceptor stem plays a role in amino acid discrimination. A direct comparison of the initial rates of aminoacylation with both isoleucine and valine reveals that isoleucine is only a 3-fold better substrate under these conditions. This level of discrimination was observed over a large concentration range (50–500 μm) of minihelix substrate (data not shown), showing that the nature of the amino acid does not modulate minihelix binding. Because the rates of Ile-AMP and Val-AMP synthesis are greater than the rate of aminoacylation of minihelix substrates, it is likely that the aminoacyl adenylates (of both isoleucine and valine) accumulate in the active site to similar levels. Under these circumstances, the rate of aminoacyl-RNA formation should be most correlated to the transfer rate of the aminoacyl group from the adenylate to the minihelix. The small difference between the isoleucylation and valylation rate of minihelixIle may represent a lack of discrimination toward the amino acid side chain in the transfer step. The ability of isoleucyl-tRNA synthetase to discriminate against valine is enhanced in the presence of tRNAIle. In charging of minihelix substrates, this discrimination is reduced to a mere 3-fold preference for isoleucine. This outcome is expected for substrates that have significantly reduced editing activity and yet have retained some charging activity. The mischarging of a minihelix reinforces the evidence that the D-loop of tRNAIle is indispensable for proper discrimination against valine (13Hale S.P. Auld D.S. Schmidt E. Schimmel P. Science. 1997; 276: 1250-1252Crossref PubMed Scopus (79) Google Scholar). To some extent this finding has been puzzling, as it is thought that IleRS makes little contact with the D-loop of tRNAIle; for example, none of the D-loop nucleotides critical for editing are protected by bound IleRS in phosphate ethylation experiments (14Nureki O. Niimi T. Muramatsu T. Kanno H. Kohno T. Florentz C. Giegé R. Yokoyama S. J. Mol. Biol. 1994; 236: 710-724Crossref PubMed Scopus (101) Google Scholar). Considering the available evidence, we propose that the effect of the D-loop is derived from its presumed role in transducing conformational information between the two domains of tRNAIle. Long range information transfer through the tRNA structure is also suggested from studies of the aminoacylation of tRNAIle by IleRS. For example, mutations in the anticodon of tRNAIle (which interacts directly with IleRS) do not affect binding but rather diminish the kcat for charging. In a model for aminoacylation of wild-type tRNAIle, binding of the correct anticodon enables conformational changes that accurately orient the acceptor terminus in the distant active site (14Nureki O. Niimi T. Muramatsu T. Kanno H. Kohno T. Florentz C. Giegé R. Yokoyama S. J. Mol. Biol. 1994; 236: 710-724Crossref PubMed Scopus (101) Google Scholar). The charging of minihelices by IleRS provides a model aminoacylation system where one or more of the discriminatory "sieves" has been attenuated. It should be noted that the notion of doublesieve discrimination is actually semantic in that editing by IleRS is known to involve two editing reactions (i.e. pre- and post-transfer). Considering the initial activation sieve, there are at least three functional sieves. In theory, discrimination against valine could involve numerous other sieves, each acting at a unique kinetic step along the aminoacylation reaction coordinate. One such step that could enhance discrimination against valine is the transfer of the aminoacyl group from the activated adenylate to the accepting 2′-OH of the RNA. However, our results suggest that this is not the case. In the aminoacylation of minihelices (where the aminoacylation rate is likely to reflect the transfer rate) the preference for isoleucine is quite small. Mutations in minihelix substrates that affect the aminoacylation rate do so independently of the amino acid side chain. At least for the Δ1 minihelixIle mutation, we obtained evidence that the mutation directly affects the transfer rate with little or no effect on binding. Although the high K mvalues (>200 μm) for these substrates make this parameter difficult to measure precisely, approximateK m values for minihelixIle and Δ1 minihelixIle are within 20% of each other (data not shown). In contrast, the kcat value for Δ1 minihelixIle is more than 3-fold higher than that for minihelixIle in charging with both isoleucine and valine. These experiments offer further evidence that transfer of the aminoacyl group from the activated adenylate to the RNA does not provide significant discrimination against valine. Several considerations have led to the hypothesis that minihelices were evolutionary precursors to modern tRNAs (37Weiner A.M. Maizels N. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7383-7387Crossref PubMed Scopus (257) Google Scholar, 38Maizels N. Weiner A.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6729-6734Crossref PubMed Scopus (168) Google Scholar, 39Schimmel P. Giegé R. Moras D. Yokoyama S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8763-8768Crossref PubMed Scopus (347) Google Scholar, 40Schimmel P. Ribas de Pouplana L. Cell. 1995; 81: 983-986Abstract Full Text PDF PubMed Scopus (155) Google Scholar) and had an origin distinct from that of the anticodon-containing domain (41Noller H.F. Gesteland R.F. Atkins J.F. The RNA World. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 137-156Google Scholar). The correlation of acceptor stem nucleotides in minihelices with the charging of specific amino acids constitutes an operational RNA code (that could have preceded the anticodon-dependent genetic code (42Rodin S. Rodin A. Ohno S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4537-4542Crossref PubMed Scopus (87) Google Scholar)) for amino acids (39Schimmel P. Giegé R. Moras D. Yokoyama S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8763-8768Crossref PubMed Scopus (347) Google Scholar). Therefore, it is interesting to wonder whether the relaxed amino acid specificity in the aminoacylation of minihelixIle reported here is indicative of more primitive aminoacylation systems in general. Perhaps during the early development of an operational RNA code aminoacylation systems with greater net catalytic activity had a selective advantage over those with lower, yet more specific catalytic activity. Subsequently, as aminoacylation systems became more robust, specificity could have become a greater selective advantage. At this point, determinants for editing may have been appended to the tRNA structure.