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Sequences Outside Recognition Sets Are Not Neutral for tRNA Aminoacylation

1998; Elsevier BV; Volume: 273; Issue: 19 Linguagem: Inglês

10.1074/jbc.273.19.11605

1083-351X

Magali Frugier, Mark Helm, Brice Felden, Richard Giegé, Catherine Florentz,

RNA Research and Splicing

Phenylalanine identity of yeast tRNAPhe is governed by five nucleotides including residues A73, G20, and the three anticodon nucleotides (Sampson et al., 1989, Science 243, 1363–1366). Analysis of in vitro transcripts derived from yeast tRNAPhe and Escherichia colitRNAAla bearing these recognition elements shows that phenylalanyl-tRNA synthetase is sensitive to additional nucleotides within the acceptor stem. Insertion of G2-C71 has dramatic negative effects in both tRNA frameworks. These effects become compensated by a second-site mutation, the insertion of the wobble G3-U70 pair, which by itself has no effect on phenylalanylation. From a mechanistic point of view, the G2-C71/G3-U70 combination is not a “classical” recognition element since its antideterminant effect is compensated for by a second-site mutation.This enlarges our understanding of tRNA identity that appears not only to be the outcome of a combination of positive and negative signals forming the so-called recognition/identity set but that is also based on the presence of nonrandom combinations of sequences elsewhere in tRNA. These sequences, we name “permissive elements,” are retained by evolution so that they do not hinder aminoacylation. Likely, no nucleotide within a tRNA is of random nature but has been selected so that a tRNA can fulfill all its functions efficiently. Phenylalanine identity of yeast tRNAPhe is governed by five nucleotides including residues A73, G20, and the three anticodon nucleotides (Sampson et al., 1989, Science 243, 1363–1366). Analysis of in vitro transcripts derived from yeast tRNAPhe and Escherichia colitRNAAla bearing these recognition elements shows that phenylalanyl-tRNA synthetase is sensitive to additional nucleotides within the acceptor stem. Insertion of G2-C71 has dramatic negative effects in both tRNA frameworks. These effects become compensated by a second-site mutation, the insertion of the wobble G3-U70 pair, which by itself has no effect on phenylalanylation. From a mechanistic point of view, the G2-C71/G3-U70 combination is not a “classical” recognition element since its antideterminant effect is compensated for by a second-site mutation. This enlarges our understanding of tRNA identity that appears not only to be the outcome of a combination of positive and negative signals forming the so-called recognition/identity set but that is also based on the presence of nonrandom combinations of sequences elsewhere in tRNA. These sequences, we name “permissive elements,” are retained by evolution so that they do not hinder aminoacylation. Likely, no nucleotide within a tRNA is of random nature but has been selected so that a tRNA can fulfill all its functions efficiently. The specificity of transfer RNA aminoacylation is a crucial step in protein synthesis. Investigations during the last years have shown that the aminoacylation identity of a tRNA is linked to the presence of specific sets of signals allowing both discrimination by cognate aminoacyl-tRNA synthetases (aaRSs), 1The abbreviations used are: aaRS, aminoacyl-tRNA synthetase; AlaRS, AspRS, PheRS, SerRS, ValRS, alanyl-, aspartyl-, phenylalanyl-, seryl-, and valyl-tRNA synthetases; DTE, diifthioerythritol. the positive elements, and rejection by noncognate synthetases, the negative elements or antideterminants (1Giegé R. Puglisi J.D. Florentz C. Prog. Nucleic Acid Res. Mol. Biol. 1993; 45: 129-206Crossref PubMed Scopus (218) Google Scholar, 2Saks M.E. Sampson J.R. Abelson J.N. Science. 1994; 263: 191-197Crossref PubMed Scopus (150) Google Scholar, 3McClain W.H. Söll D. RajBhandary U.L. tRNA: Structure, Biosynthesis, and Function. American Society of Microbiology Press, Washington, D. C.1995: 335-347Google Scholar). The completeness of a set of positive elements has generally been tested by co-transplantation of the corresponding nucleotides into one or several noncognate host tRNAs that acquire the new aminoacylation properties. In several instances, this approach allowed detection of special requirements for the optimal expression of a given aminoacylation identity set within a host tRNA. Thus, minor elements and conformational features were shown to contribute to aminoacylation identities (e.g. Refs.4McClain W. Foss K. Jenkins R.A. Schneider J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9272-9276Crossref PubMed Scopus (52) Google Scholar, 5Francklyn C. Musier-Forsyth K. Schimmel P. Eur. J. Biochem. 1992; 206: 315-321Crossref PubMed Scopus (40) Google Scholar, 6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar, 7Hou Y.-M. Westhof E. Giegé R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6776-6780Crossref PubMed Scopus (108) Google Scholar, 8Becker H.D. Giegé R. Kern D. Biochemistry. 1996; 35: 7447-7458Crossref PubMed Scopus (66) Google Scholar). Recognition elements required for phenylalanylation of yeast tRNAPhe were defined in the pioneering work of Uhlenbeck and co-workers (9Sampson J.R. Uhlenbeck O.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1033-1037Crossref PubMed Scopus (613) Google Scholar, 10Sampson J.R. DiRenzo A.B. Behlen L.S. Uhlenbeck O.C. Science. 1989; 24: 1363-1366Crossref Scopus (148) Google Scholar) as a set of five major elements. These elements correspond to G20, G34, A35, A36, and A73, and their competence to confer phenylalanine (Phe) identity was first demonstrated by transplantation into four host tRNAs that all acquired optimal phenylalanylation capacities (10Sampson J.R. DiRenzo A.B. Behlen L.S. Uhlenbeck O.C. Science. 1989; 24: 1363-1366Crossref Scopus (148) Google Scholar). Nucleotides involved in tertiary interactions were shown not to contribute to identity by a direct effect (11Sampson J. DiRenzo A.B. Behlen L.S. Uhlenbeck O.C. Biochemistry. 1990; 29: 2523-2532Crossref PubMed Scopus (93) Google Scholar). Alternatively, expression of Phe identity in the yeast tRNAAsp context has revealed that PheRS is sensitive to fine local structural features, such as the D-loop and variable region structures (12Perret V. Florentz C. Puglisi J.D. Giegé R. J. Mol. Biol. 1992; 226: 323-333Crossref PubMed Scopus (47) Google Scholar). Finally, in neither study based on sequence comparisons of natural or engineered Phe accepting species, nucleotides within the acceptor stem helix were found important for specificity. In a previous work, we have been able to create a chimeric tRNA, efficiently recognized and aminoacylated at once by three different aminoacyl-tRNA synthetases including yeast PheRS (6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar), and found that effective phenylalanylation of this tRNA was dependent, among other features, on the sequence of base pair 2–71 within the acceptor stem. Engineering of a tRNA with multiple specificities was based on the synthesis of a chimeric tRNAAsp containing the recognition sets for yeast PheRS (the five residues listed above), forEscherichia coli AlaRS (the G3-U70 base pair,e.g. Refs. 13Hou Y.-M. Schimmel P. Nature. 1988; 333: 140-145Crossref PubMed Scopus (513) Google Scholar and 14McClain W.H. Foss K. Science. 1988; 240: 793-796Crossref PubMed Scopus (292) Google Scholar), and for yeast ValRS (A73 and A35; Ref. 15Florentz C. Dreher T.W. Rudinger J. Giegé R. Eur. J. Biochem. 1991; 195: 229-234Crossref PubMed Scopus (31) Google Scholar). Notice that the valine identity residues A73 and A35 are common to the Phe recognition set. Simultaneous optimization of alanylation and phenylalanylation efficiencies could be achieved by insertion of specific structural features (the length of the α- and β-domains within the D-loop shaped to 4 and 2 nucleotides and the length of the variable region extended to 5 nucleotides) and mutation of base pair 2–71 in the amino acid acceptor stem from C-G to G-C (6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar). Whereas the structural changes introduced were directed by already established yeast PheRS requirements (11Sampson J. DiRenzo A.B. Behlen L.S. Uhlenbeck O.C. Biochemistry. 1990; 29: 2523-2532Crossref PubMed Scopus (93) Google Scholar, 12Perret V. Florentz C. Puglisi J.D. Giegé R. J. Mol. Biol. 1992; 226: 323-333Crossref PubMed Scopus (47) Google Scholar), replacement of base pair C2-G71 by G2-C71 in the chimeric tRNAAsp transcript was guided by our present understanding of E. colitRNAAla identity. Indeed, this base pair is important for optimal alanine identity expression (5Francklyn C. Musier-Forsyth K. Schimmel P. Eur. J. Biochem. 1992; 206: 315-321Crossref PubMed Scopus (40) Google Scholar, 16Shi J.P. Francklyn C. Hill K. Schimmel P. Biochemistry. 1990; 29: 3621-3626Crossref PubMed Scopus (57) Google Scholar, 17McClain W. Foss K. Jenkins R.A. Schneider J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6147-6151Crossref PubMed Scopus (55) Google Scholar) but was not expected to be of any influence on phenylalanylation (10Sampson J.R. DiRenzo A.B. Behlen L.S. Uhlenbeck O.C. Science. 1989; 24: 1363-1366Crossref Scopus (148) Google Scholar, 18Sampson J.R. Behlen L.S. DiRenzo A.B. Uhlenbeck O.C. Biochemistry. 1992; 31: 4164-4167Google Scholar). Enhancement of phenylalanylation activity by insertion of a G2-C71 base pair was effective in three different structural contexts, all containing the G3-U70 base pair required for efficient alanylation (6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar). Here, we basically investigate the role of base pairs 2–71 and 3–70 in Phe identity in yeast tRNAPhe as well as in the frameworks of yeast tRNAAsp and E. colitRNAAla. The kinetic data collected for various T7 transcripts demonstrate that these base pairs are involved in an optimal expression of the yeast tRNAPhe recognition set. Several possible roles played by these nucleotides are discussed. They do clearly not behave as positive recognition elements as commonly defined in the field. The new outcome of our studies is that the sequence of a tRNA, apart from the recognition elements and the consensus nucleotides involved in the establishment of the three-dimensional structure, is not random for aminoacylation. Some combinations are tolerated, others are not. Thus, specificity is linked to the set of positive and negative recognition elements as well as to an adequate sequence combination within the remaining domains of the tRNA. Oligonucleotides were synthesized on an Applied Biosystems 381 DNA synthesizer using the phosphoramidite method and purified by HPLC on a Nucleosyl 125–5-C18 column (Bischoff Chromatography, Zymark-France, Paris). l-[3H] phenylalanine (9.6 × 1011 Bq/mol) was from Amersham France (Les Ulis). Yeast PheRS was a gift of M. Baltzinger (Strasbourg). T7 RNA polymerase was purified according to method as described previously (19Wyatt J.R. Chastain M. Puglisi J.D. BioTechniques. 1991; 11: 764-769PubMed Google Scholar). Restriction enzymes (BstN1,HindIII, and BamHI) and T4 polynucleotide kinase were from New England Biolabs (Beverly, MA). T4 DNA ligase was from Boehringer Mannheim (Meylan, France). All tRNAs used in this work have been obtained by in vitro transcription of synthetic genes. Each of these genes corresponds to the T7 RNA polymerase promoter region directly upstream of the tRNA sequence. The tRNA genes were constructed and cloned into plasmid pUC 119 linearized at BamHI and HindIII sites according to established methods (20Perret V. Florentz C. Giegé R. FEBS Lett. 1990; 270: 4-8Crossref PubMed Scopus (14) Google Scholar). Tg1 cells were transformed. ABstN1 site coincidental with the 3′-end of the tRNA sequences allows synthesis of tRNAs ending with the expected CCA sequence. Experimental procedures were described previously (20Perret V. Florentz C. Giegé R. FEBS Lett. 1990; 270: 4-8Crossref PubMed Scopus (14) Google Scholar).In vitro preparation and purification of transcripts was performed according to established procedures (6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar). Concentration of stock solutions of transcripts have been determined by absorbency measurements at 260 nm. The primary structure of variants has been checked, in particular for the 5′-end G-rich regions where the sequences were carefully verified by appropriate sequencing methods (21Brownlee G.G. Work T.S. Work E. Determination of Sequences in RNA. Laboratory Techniques in Biochemistry and Molecular Biology. North Holland Publishing Company, Amsterdam1972Google Scholar, 22Donnis-Keller H. Maxam A.M. Gilbert W. Nucleic Acids Res. 1977; 4: 2527-2538Crossref PubMed Scopus (1040) Google Scholar). The global folding of the variant transcripts was shown to be the same as that of wild-type tRNAPhe transcript, as demonstrated by structural mapping of the RNAs by lead according to procedures described previously (23Krzyzosiak W.J. Marciniec T. Wiewiorowsky M. Romby P. Ebel J.-P. Giegé R. Biochemistry. 1988; 27: 5771-5777Crossref PubMed Scopus (102) Google Scholar, 24Behlen L.S. Sampson J.R. DiRenzo A.B. Uhlenbeck O.C. Biochemistry. 1990; 29: 2515-2523Crossref PubMed Scopus (154) Google Scholar). Aminoacylation reactions of transcripts derived from tRNAAsp have been performed as described (6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar) in a medium containing 25 mm Tris-HCl, pH 7.5, 7.5 mm MgCl2, 0.5 mm ATP, 0.1 mg/ml bovine serum albumin, 50 μm[3H]-labeled phenylalanine, and adequate amounts of tRNA transcript and yeast PheRS. Aminoacylation reactions of transcripts derived from tRNAPhe and tRNAAlahave been performed in a medium containing 30 mm HEPES, pH 7.4, 15 mm MgCl2, 12 mm ATP, 30 mm KCl, 4 mm DTE, 50 μm[3H]-labeled phenylalanine, tRNA transcript, and yeast PheRS (10Sampson J.R. DiRenzo A.B. Behlen L.S. Uhlenbeck O.C. Science. 1989; 24: 1363-1366Crossref Scopus (148) Google Scholar). Before aminoacylation, transcripts were renatured by heating at 65 °C for 90 s and slow cooling to room temperature. Assays were performed in the conventional way (25Perret V. Garcia A. Grosjean H. Ebel J.-P. Florentz C. Giegé R. Nature. 1990; 344: 787-789Crossref PubMed Scopus (178) Google Scholar) with incubation at 30 °C. The kinetic constants were derived from Lineweaver-Burk plots. Since the concentration of amino acids is subsaturating, only apparent kinetic parameters are given. They represent an average of at least two independent experiments. Functional properties of mutants are expressed as catalytic efficiencies of phenylalanylation byk cat over K m ratios. For easier comparisons, these ratios are also normalized with regard to the wild-type molecule. All experiments performed in our previous work (6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar) leading to the discovery of a positive effect of a G2-C71 base pair on phenylalanylation were done in the tRNAAsp context. These transcripts contained systematically a G3-U70 base pair, necessary for an efficient concomitant alanylation (6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar). Influence of this last base pair on the chimeric tRNAAsp phenylalanylation activity was tested by comparison with the activity of two types of tRNAAsp transcripts bearing the five primary Phe recognition nucleotides and various combinations at positions 2–71 and 3–70 (Fig.1). Variant A has the basic sequence of tRNAAsp, the structural framework of tRNAAsp, and the five phenylalanine identity nucleotides (12Perret V. Florentz C. Puglisi J.D. Giegé R. J. Mol. Biol. 1992; 226: 323-333Crossref PubMed Scopus (47) Google Scholar). Variant B represents the corresponding transcript, containing a G3-U70 base pair. The kinetic parameters of these transcripts (TableI) show that the presence of base pair G3-U70 has only a very limited effect (at most, a 5-fold decrease on aminoacylation efficiency). In this context, when C2-G71 is replaced with G2-C71 (variant C), phenylalanylation efficiency is increased 60-fold. About the same effects were obtained when the structural framework of the host tRNAAsp was engineered to mimic the structural characteristics of tRNAPhe (in the D-loop and variable region; Ref. 6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar). This first set of data led us to suspect the positive effect of the combination of G2-C71/G3-U70 base pairs on phenylalanylation.Table IKinetic parameters for phenylalanylation by yeast PheRS of several transcripts bearing phenylalanine recognition nucleotidestRNA transcriptsBase pairs at position 2–71 & 3–70k cat(s −1)K m (nm)k cat/K m (×1000)k cat/K m (relative)L (-fold)Transcripts derived from yeast tRNAAspaPhenylalanylation conditions according to Ref. 6. DCG /GC (wt)23006.6611 ACG /CG0.17170.140.02148 BCG /GU0.0621850.0270.004243 CGC /GU2.716251.660.254Transcripts derived from yeast tRNAPhebPhenylalanylation conditions according to Ref. 10. DCG /GC (wt)6.347013.411 EAU /GC4.210004.20.313 FGC /GC0.08100000.0080.00061675 GCG /GU8.0629002.80.215 HGC /GU141400100.751Transcripts derived from E. coli tRNAAlabPhenylalanylation conditions according to Ref. 10. ICG /GC (wt)0.6240000.1550.0186 JGC /GC0.05586000.00640.00052096 KCG /GU5.7540014.41.071 LGC /GU0.88700.920.06815a Phenylalanylation conditions according to Ref. 6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar.b Phenylalanylation conditions according to Ref. 10Sampson J.R. DiRenzo A.B. Behlen L.S. Uhlenbeck O.C. Science. 1989; 24: 1363-1366Crossref Scopus (148) Google Scholar. Open table in a new tab In order to study the role of base pairs 2–71 and 3–70 on phenylalanylation in the cognate natural tRNA framework, a series of mutants of yeast tRNAPhe presenting variations in their sequence have been prepared by in vitro transcription (Fig. 1). The wild-type tRNAPhe transcript presents the sequences C2-G71 and G3-C70, and is referred as molecule D in this work (Fig. 1). Four mutants were designed. Variants E and F differ from wild-type tRNAPhe only at the level of the second base pair, where C-G was replaced with A-U (variant E) or G-C (variant F). Variant G differs from wild-type at the third base pair by the presence of a G-U pair. This mutant was constructed to test the role of the alanine identity element G3-U70 alone in phenylalanylation. Finally, the last mutant (variant H) contains two concomitant substitutions in the acceptor stem, namely G2-C71 and G3-U70. Kinetic parameters characterizing the phenylalanylation capacity of these mutants are summarized in Fig. 1 and Table I. A dramatic effect is observed by inversion of base pair 2–71 from C-G to G-C (variant F), with of more than 1600-fold loss of phenylalanylation efficiency (in comparison to wild-type transcript D). This large decrease is due to a large drop of the rate constant (78-fold) and an increment ofK m by a factor 21 (Table I). Interestingly, the presence of an A-U base pair at this same position (variant E) has no effect on phenylalanylation efficiency. Conversion of base pair 3–70 from G-C to G-U in the tRNAPhe context (variant G), has only a moderate effect on phenylalanylation (5-fold decrease). The K m is increased about 6-fold and k cat is not quite affected (Table I). Interestingly, the double mutant (variant H) behaves like the wild-type transcript although it contains a mutation (G2-C71) that affects the aminoacylation in variant F in a strong negative manner. The aminoacylation rate of this mutant is two times better than for the wild-type tRNA, but K m is increased 3-fold. Thus, as in the tRNAAsp context, the presence of base pairs G2-C71/G3-U70 is positively recognized by PheRS in the tRNAPhe context. Effect of base pairs 2–71 and 3–70 on phenylalanylation has been further tested within a third tRNA context, namely E. coli tRNAAla. Sequences of the tested transcripts are displayed in Fig. 1. Note that these transcripts share the same fine structural characteristics as tRNAPhe in terms of d-loop and variable region organizations, namely the same length of the α and β regions in thed-loop and the same length of the variable region (v = 5). Since constant G18 and G19 residues in thed-loop make long range interactions with the T-loop, in particular the G19-C56 Watson-Crick pair, and that variable region residues are structurally related with d-loop and stem residues (e.g. the G15-C48 Levitt pair and the C13-G22-G46 triple), it can be concluded that the core of both tRNAPheand tRNAAla transcripts are similar and consequently that the two tRNAs present the same overall three-dimensional structure (1Giegé R. Puglisi J.D. Florentz C. Prog. Nucleic Acid Res. Mol. Biol. 1993; 45: 129-206Crossref PubMed Scopus (218) Google Scholar,6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar). Variant I, a tRNAAla with the Phe recognition set and the same sequence as wild-type tRNAPhe at positions 2–71 and 3–70, is 85-fold less well aminoacylated than the reference tRNAPhe transcript. Both k cat andK m are about 10-fold lower. Interestingly, in variant J, as in tRNAPhe, a G2-C71/G3-C70 combination of nucleotides has a dramatic negative consequence on phenylalanylation with a loss in aminoacylation efficiency of 2100-fold. Finally, sequence combinations C2-G71/G3-U70 or G2-C71/G3-U70 lead to efficient phenylalanylation of tRNAAla-derived tRNAs (L = 1 and L = 14, respectively, for variants K and L), as is the case in the tRNAPhe context (variant H). Calculation of thermodynamic stabilities of RNA helices was according to Turner et al.(26Turner D.H. Sugimoto N. Freier S.M. Annu. Rev. Biophys. Biophys. Chem. 1988; 17: 167-193Crossref PubMed Scopus (582) Google Scholar). In the presence of a G3-C70 pair, the free energy of the tRNAPhe accepting helix is between 2 and 3 kcal/mol lower than in the presence of G3-U70. Indeed, in the case of the tRNAPhe framework, the combination G2-C71/G3-C70 confers a free energy of −7.1 kcal/mol, whereas G2-C71/G3-U70 confers only −5.4 kcal/mol. Similarly, in the tRNAAla framework, a G-C/G-C combination leads to −8.7 kcal/mol, whereas a G-C/G-U combination leads only to −5.7 kcal/mol. First, we recall that the tRNAs investigated here possess the foreseen sequences and fold correctly as verified by sequence analysis and structural mapping with lead (data not shown). Further, these molecules, aside from containing the Phe recognition elements, possess all the same sequences at their amino acid-accepting extremities, namely a G1-C72 base pair extended by a 3′ A73CCA-end. In what follows, and for the sake of simplicity, we assume that the variations in their phenylalanylation properties are essentially due to the nucleotide combinations at positions 2–71 and 3–70, although we are aware that additional effects can occur (see below). To facilitate discussion, data are summarized in TableII. Variants are classified as a function of their decreasing ability to be phenylalanylated (increasingL-values) and are divided into six groups (a to f) on the basis of the nature of their 2–71 and 3–70 base pairs, and individual kinetic parameters are normalized so that relative contributions ofk cat and K m to Lcan be easily compared. Several features become immediately apparent. Similar L-values can result from different combinations ofk cat and K m . This is for instance, the case when comparing the phenylalanylation capacities of variants H and K. The normalized k cat andK m show that efficient variants (L< 5) behave phenomenologically either as wild-type tRNAPhe(variants K and E) or are charged in a mechanism where their binding to PheRS is decreased (normalized K m > 1) andk cat improved (variants H, C, and G). Interestingly, in variants with impaired aminoacylation capacity (L > 5), the contribution ofk cat is preponderant, except for variant I.Table IIRanking of tRNA variants as a function of their efficiency to be phenylalanylated and comparison of the relative contributions of k cat and K m in chargingtRNA frameworkName of variants & base pairs 2–71/3–70L (-fold)Normalized values of k cat and K m for L = 1abcdefC-G G-CG-C G-UG-C G-CC-G G-UA-U G-CC-G C-G(k cat)N(K m )NPhe—————11.001.00Phe—H————10.392.59Ala———K——11.140.88Phe————E—30.851.19Asp—C————40.372.70Phe———G——50.362.81Ala—L————152.040.49Asp—————A482.870.35AlaI—————861.100.92Asp———B——2432.140.47Phe——F———16751.930.51Ala——J———20962.500.40 Open table in a new tab Among the 11 tRNA variants possessing the basic Phe recognition set and mutations at base pairs 2–71 and/or 3–70, two mutants with a G2-C71 base pair have drastically decreased aminoacylation efficiencies as compared with wild-type tRNAPhe (variants F and J). The magnitude of the effects, with l-values of 1675 to 2100, is much greater than that observed after mutation of the “classical” Phe recognition elements where losses varied from 10- to 260-fold (10Sampson J.R. DiRenzo A.B. Behlen L.S. Uhlenbeck O.C. Science. 1989; 24: 1363-1366Crossref Scopus (148) Google Scholar, 18Sampson J.R. Behlen L.S. DiRenzo A.B. Uhlenbeck O.C. Biochemistry. 1992; 31: 4164-4167Google Scholar). But, analysis of tRNA variants presenting a G-C/G-U combination at positions 2–71/3–70, shows that the dramatic negative effect brought by G2-C71 in the tRNAPhe and tRNAAla contexts (variants F and J), is compensated by the G3-U70 base pair (variants H, C, and L). Notably, the G2-C71/G3-U70 combination in the tRNAAspframework has a positive effect (variant C). According to the current view, tRNA aminoacylations are ensured by a limited number of nucleotides acting as positive signals (1Giegé R. Puglisi J.D. Florentz C. Prog. Nucleic Acid Res. Mol. Biol. 1993; 45: 129-206Crossref PubMed Scopus (218) Google Scholar, 2Saks M.E. Sampson J.R. Abelson J.N. Science. 1994; 263: 191-197Crossref PubMed Scopus (150) Google Scholar, 3McClain W.H. Söll D. RajBhandary U.L. tRNA: Structure, Biosynthesis, and Function. American Society of Microbiology Press, Washington, D. C.1995: 335-347Google Scholar). They are presented within optimal structural scaffolds and are generally in direct contact with specific amino acids of synthetases. This definition has two implications. First, mutations at recognition positions should lead to strong losses in aminoacylation efficiency, due to the disappearance of proper hydrogen bonds. Second, transplantation of recognition elements into host tRNA frameworks should be sufficient for acquisition of the corresponding aminoacylation identity by the chimeric tRNAs. The properties of the C2-G71 base pair are not explained by the above scheme. Indeed, our results, combined with those of Uhlenbeck's laboratory, suggest that PheRS makes no distinction between a U-A, A-U, or C-G base pair at position 2–71, and G-C is the only pair to considerably decrease aminoacylation efficiency of the tRNAs chargeable with phenylalanine. Moreover, C2-G71 is not necessary to confer an efficient Phe identity to a host tRNA. Thus, C2-G71 is not a positive identity element. Alternatively, mutation of a neutral element, by definition an element that does not belong to the recognition set, can also lead to a dramatic loss in aminoacylation efficiency. This is the case if this “neutral” element is replaced by a negative element that can act by introducing a repelling chemical group toward the synthetase or an unfavorable structural context, hindering the correct local or long-range positioning of the substrate. Thus G2-C71 has to be considered as a “negative” element. However, it is not clear if this base pair is in direct contact with the synthetase, although footprinting of natural fully modified tRNAPhe with yeast PheRS showed protection of phosphates 69 and 70 against ethylnitrosourea alkylation (27Romby P. Moras D. Bergdoll M. Dumas P. Vlassov V.V. Westhof E. Ebel J.- P. Giegé R. J. Mol. Biol. 1985; 184: 455-471Crossref PubMed Scopus (110) Google Scholar). The recent crystal structure of the tRNAPhe/PheRS complex from Thermus thermophilus(28Goldgur Y. Mosyak L. Reshetnikova L. Ankilova V. Lavrik O. Khodyreva S. Safro M. Structure. 1997; 5: 59-68Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) indicates that there is no contact between the enzyme and base pairs 2–71 and 3–70 of the tRNA. Expression of the information carried by base pair G2-C71 is largely dependent on the sequence of base pair 3–70. Its negative effect, observed when position 3–70 is a G-C pair (variants F and J), is balanced by the presence of a G3-U70 pair (variants H and L). Thus, a given base pair affect phenylalanylation differently according to the tRNA framework into which it is embedded. Here again, G3-U70 cannot by itself be considered as a positive recognition signal for yeast PheRS. This conclusion is in line with the great sequence variability of base pair 3–70 among the host tRNAs into which the Phe recognition set has been transplanted (C-G in S. pombe tRNAPhe; G-C inE. coli, yeast, and wheat germ tRNAPhe and yeast tRNATyr; U-A in yeast tRNAMet; and G-U in yeast tRNAArg) (10Sampson J.R. DiRenzo A.B. Behlen L.S. Uhlenbeck O.C. Science. 1989; 24: 1363-1366Crossref Scopus (148) Google Scholar). The positive effect brought by the G2-C71/G3-U70 combination may be related to the structural characteristics of the G-U pair that decrease the stability of the acceptor stem in comparison with a stem with standard Watson-Crick pairings. We recall that G-U base pairs are important in protein-RNA interactions in general, and in tRNA recognition in particular. For instance, the G3-U70 pair in tRNAAla is the major alanine identity element for AlaRS andin E. coli is involved in a subtle recognition process by the synthetase (4McClain W. Foss K. Jenkins R.A. Schneider J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9272-9276Crossref PubMed Scopus (52) Google Scholar, 29Musier-Forsyth K. Usman N. Scaringe S. Doudna J. Green R. Schimmel P. Science. 1991; 253: 784-786Crossref PubMed Scopus (135) Google Scholar, 30Musier-Forsyth K. Schimmel P. Nature. 1992; 357: 513-515Crossref PubMed Scopus (105) Google Scholar, 31Musier-Forsyth K. Shi J.-P. Henderson B. Bald R. Fürste J.P. Erdmann V.A. Schimmel P. J. Am. Chem. Soc. 1995; 117: 7253-7254Crossref Scopus (32) Google Scholar, 32Gabriel K. Schneider J. McClain W.H. Science. 1996; 271: 195-197Crossref PubMed Scopus (90) Google Scholar). In addition to the presence of the exocyclic amino group in the minor groove of the RNA helix, a G-U pair decreases the helix stability as compared with its G-C equivalent since it contains only two hydrogen bonds (33Ladner J.E. Jack A. Robertus J.D. Brown R.S. Rhodes D. Clark B.F.C. Klug A. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 4414-4418Crossref PubMed Scopus (236) Google Scholar, 34Rhodes D. Eur. J. Biochem. 1977; 81: 91-101Crossref PubMed Scopus (35) Google Scholar, 35Aboul-ela F. Koh D. Tinoco Jr., I. Martin F.H. Nucleic Acids Res. 1985; 13: 4811-4824Crossref PubMed Scopus (412) Google Scholar). Unlike a G-C pair, a G-U pair was shown, by NMR, to induce a variation in the local helix geometry (36Allain F.H.T. Varani G. J. Mol. Biol. 1995; 250: 333-353Crossref PubMed Scopus (285) Google Scholar, 37Limmer S. Reif B. Ott G. Arnold L. Sprinzl M. FEBS Lett. 1996; 385: 15-20Crossref PubMed Scopus (44) Google Scholar, 38Ramos A. Varani G. Nucleic Acids Res. 1997; 25: 2083-2090Crossref PubMed Scopus (86) Google Scholar). For example, in an RNA helix mimicking the tRNAAla acceptor stem, the G-U pair displaces nucleotide C71 and reduces the stacking of the four unpaired nucleotides at the 3′-extremity of the helix (37Limmer S. Reif B. Ott G. Arnold L. Sprinzl M. FEBS Lett. 1996; 385: 15-20Crossref PubMed Scopus (44) Google Scholar). The yeast tRNAAsp anticodon helix is another example where a noncanonical G-U pair plays an role in the interaction process with its cognate synthetase by destabilizing an RNA helix. In this case, the G30-U40 pair was shown to be the site of a kink (39Westhof E. Dumas P. Moras D. J. Mol. Biol. 1985; 184: 119-145Crossref PubMed Scopus (427) Google Scholar) that allows adaptation of the anticodon nucleotides with the AspRS anticodon binding domain (40Cavarelli J. Rees B. Ruff M. Poterszman A. Thierry J.-C. Moras D. Structural Tools for the Analysis of Protein-Nucleic Acid Complexes. Birkhäuser Verlag, Basel, Switzerland1992: 287-298Google Scholar). Within these lines, we suggest that introduction of a G3-U70 pair into tRNAs inactive for phenylalanylation brings sufficient flexibility into the acceptor stem to allow a fruitful adaptation of the tRNA to PheRS. Similar interpretation has been given to explain the role of G3-U70 in Ala identity in vivo (32Gabriel K. Schneider J. McClain W.H. Science. 1996; 271: 195-197Crossref PubMed Scopus (90) Google Scholar), although in that case other in vitro data argue that direct recognition of this pair by the synthetase is more important than helical distortion (41Beuning P.J. Yang F. Schimmel P. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10150-10154Crossref PubMed Scopus (40) Google Scholar). Calculations of the thermodynamic stability (26Turner D.H. Sugimoto N. Freier S.M. Annu. Rev. Biophys. Biophys. Chem. 1988; 17: 167-193Crossref PubMed Scopus (582) Google Scholar) of the acceptor helix part estimated for the top four base pairs of the series of variants investigated here argue in this way. The G3-U70 base pair considerably decreases the stability of the acceptor stem, and the flexibility brought by the G2-C71/G3-U70 combination may be sufficient to recover the correct position of the CCA-accepting end. In contrast, when the combination G2-C71/G3-C70 is present, the stability of the acceptor stem is increased, and thus its optimal adaptation on the catalytic domain of the synthetase is prevented. The same conclusion was reached by Saks and Sampson (42Saks M.E. Sampson J.R. EMBO J. 1996; 15: 2843-2849Crossref PubMed Scopus (68) Google Scholar), who observed that base pair 3–70 in the acceptor stem of E. coli tRNASer, which does not contact directly the enzyme, can affect interactions between the neighboring base pairs and SerRS. In this case also, the phenomenon seems to correlate with a decreased flexibility of the acceptor stem, and base pair 3–70 was quoted in this case, a “cryptic” recognition element. Involvement of the presently discussed elements at positions 2–70 and 3–71 escaped previous studies although several transplantation experiments were performed. Here, a mutational analysis dictated by serendipitous observations during identity engineering studies (6Frugier M. Florentz C. Schimmel P. Giegé R. Biochemistry. 1993; 32: 14053-14061Crossref PubMed Scopus (30) Google Scholar), has revealed that tRNA nucleotides outside the already reported PheRS recognition set (9Sampson J.R. Uhlenbeck O.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1033-1037Crossref PubMed Scopus (613) Google Scholar,10Sampson J.R. DiRenzo A.B. Behlen L.S. Uhlenbeck O.C. Science. 1989; 24: 1363-1366Crossref Scopus (148) Google Scholar) can be of great influence on the expression of Phe identity. This analysis has in particular shown that PheRS accommodates equally well at least three different base pairs at position 2–71. In addition, the present experiments show that PheRS is sensitive to base pair combinations at positions 2–71 and 3–70 in host tRNAs embedding the canonical Phe recognition elements. In particular, the presence of two consecutive G-C pairs at position 2–71 and 3–70 considerably reduces phenylalanylation.