Conversion of aminoacylation specificity from tRNA to tRNA in vitro

The discrimination mechanism between tRNA" and tRNA* was studied using various in vitro transcripts of E. coli tRNATyr variants. The insertion of only two nucleotides into the variable stem of tRNAv generates serine charging activity. The acceptor activities of some of the tRNA? mutants with insertions in the long variable arm were enhanced by changes in nucleotides at positions 9 and/or 20B, which are possible elements for dictating the orientation of the long variable arm. These findings suggest that the long variable arm is involved in recognition by seryltRNA synthetase in spite of sequence and length variations shown within tRNA" isoacceptors, and eventually serves as a determinant for selection from other tRNA species. Changing the anticodon from GUA to the serine anticodon GGA resulted in a marked decrease in tyrosine charging activity, but this mutant did not show any serine charging activity. The discriminator base, the fourth base from the 3' end of tRNA, was also Important for aminoacylation with tyrosine. Complete specificity change In vitro was facilitated by insertion of three nucleotides into the variable arm plus two nucleotide changes at positions 9 and 73. INTRODUCTION Accurate discrimination of tRNAs by cognate aminoacyl-tRNA synthetases (ARSs) is required for translational fidelity. Biochemical and genetic studies have provided much information concerning tRNA acceptor identity (1-4). In many cases, the anticodon has been identified as a major identity determinant (5-10). However, the anticodon of tRNA" is apparently not involved in recognition by seryl-tRNA synthetase, since there is no conservation of anticodon bases among its isoacceptors associated with the presence of the two different types of serine codons, UCN and AGC/U (11, 12). Procaryotic tRNA", tRNA^ and tRNAy, classified as class II tRNAs, all possess a long variable arm composed of more than ten nucleotides (12). The long arm of tRNA" shows variation in both length and sequence within the isoacceptors, implying that the arm is also not involved in recognition (11 — 13). Thus, the question arises how accurate the discrimination between class II tRNAs occurs. The discriminator base, the fourth base from the 3' end of tRNA (N73), often serves as an identity element (14-18). Both G73 in tRNA", A73 in tRNA^" and A73 in tRNA *' are phylogenetically well conserved, implying an important role of this base for each tRNA in aminoacylation (12). Normanly et al. have demonstrated that a leucine-specific suppressor tRNA acquired serine specificity in vivo by a change of twelve bases, comprising the discriminator base, five bases in the acceptor stem, one base pair in the D-stem and four bases in the D-loop including alteration of the position of the GigG|9 sequence (19). However, the resulting molecule was a weak suppressor, indicating that these twelve bases do not cover a sufficient number of tRNA" identity elements. Moreover, it is difficult to explain the discrimination between tRNA and tRNA>' only in terms of the sequences of the acceptor stem and D-arm, since there is a close resemblance between the two tRNAs in these regions including the position of the G)8G|9 sequence. To gain a better understanding of this problem, the discrimination mechanism between the two tRNAs requires clarification. For this purpose, we searched for the key regions on both tRNA molecules using various in vitro transcripts of E. coli tRNAy variants. MATERIALS AND METHODS Preparation of template DNAs and in vitro transcripts Synthetic DNA oligomers carrying the T7 promoter and tRNA genes were ligated into pUC19 and transformed into E. coli strain JM109 (15, 16, 20). The template DNA sequences were confirmed by the dideoxy sequencing method (21). Each template DNA of the discriminator base-substituted mutant was prepared from the plasmid carrying the normal tRNA sequence and two synthetic primers by mutation with the polymerase chain reaction method (22). Transcripts of the tRNA genes were prepared in a reaction mixture containing 40 mM Tris-HCl (pH 8.1), 5 mM dithiothreitol, 2 mM spermidine, 10 mM MgCl2, bovine serum albumin (50 /ig/ml), 2.0 mM each NTP, 20 mM 5' GMP, BstNI* To whom correspondence should be addressed 6816 Nucleic Acids Research, Vol. 18, No. 23 digested template DNA (0.2 mg/ml), 2 units of inorganic pyrophosphatase (Sigma) and pure T7 RNA polymerase (50 /xg/ml) (15, 20, 23). The transcripts were purified by denaturating 20% polyacrylamide gel electrophoresis. Aminoacylation assay The aminoacylation reaction was carried out at 37°C in 40 ml of reaction mixture containing 60 mM Tris-HCl (pH 7.5), 10 rnM magnesium chloride, 2 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 2.5 mM ATP, 10 /xM L-[U-C]tyrosine (486 mCi/mmol) or 35 /iM L-[U-C]serine (141 mCi/mmol), 0.2—3.0 /iM transcript RNA and various concentrations of TyrRS or SerRS partially purified from E. coli strain A 19. Native tRNAy2 (1500 pmol/A26o) and tRNA**, (1300 pmol/A260) were purchased from Subriden RNA. RESULTS Acquisition of serine charging activity by insertions into the long variable arm of tRNA In each of the three class II tRNA species, conserved bases are commonly present (12), as shown in Figure 1. Comparison of the three cloverleaves shows that differences among the three tRNA species are present in the variable region. There are a few unpaired nucleotides between the possible stem of the variable arm and the base at position 48. The number of these nucleotides is zero in tRNA", one in tRNA^ (position 47J) and two in tRNAy(U47GU47H). We consider this difference as a possible cue for understanding the discrimination between the three species. We constructed several unmodified tRNA variants generated by T7 RNA transcription (Figure 2). In order to convert the variable arm of tRNA^ to the tRNA" type, two adenosine residues (A44 |A442) were inserted between C44 and G45. Surprisingly, this mutant transcript (tRNA5"3) showed a significant level of serine charging activity. The Vmax/Km was lower by three orders of magnitude than that for the transcript with a normal tRNA" sequence (Table 1). The U47GU47H deletion mutant (tRNA2), also possessing a variable arm of the tRNA" type, showed no detectable serine charging activity. These data indicated involvement of the arm length. The only bases that are absolutely conserved within the five tRNA" isoacceptors, but never present in the other two tRNA species, are D20, G ^ , G47C and G-̂ (indicated by arrows in Figure 1). Therefore, to supply the conserved base in the variable arm (G47C in tRNA "), we prepared an additional single-nucleotide (G471) insertion mutant (tRNA y4). However, the effect of the insertion was negligible. These observations indicate that the long variable arm is involved in recognition by seryl-tRNA synthetase (SerRS). All mutations within the long arm affected the tyrosine charging activity (tRNAs^ 2, 3 and 4), suggesting that the arm is also required for recognition by tyrosyl-tRNA synthetase (TyrRS). Substitutions at positions 20B and 9 Substitution of G20B for A20B in tRNA^G produced little effect on serine charging activity (tRNAy5), but a 10-fold enhancement of Vmax/Km was obtained by introduction of a simultaneous G47, insertion (tRNA ^). This suggests that the base is not a recognition element, but is required for the tertiary folding necessary for the recognition. The serine charging activity of tRNA>"3 was increased by a factor of four in Vmax/Km by substitution of G9 for U9 (tRNA *'?). The activity was greatly enhanced by additional G47, insertion (tRNA ^), whereas the degree of enhancement was lower when A471 was inserted simultaneously instead of G47il (tRNA ^). Both substitutions at positions 20B and 9 showed no significant effect on tyrosine charging activity (tRNAs>' 3 and 5, 4 and 6, 3 and 7, and 4 and 8). Substitutions of the anticodon and the discriminator base U as the second letter of the anticodon is a feature distinguishing tRNA from the other two class II tRNAs (Figure 1). Changing the anticodon from GUA to the serine anticodon GGA caused about a 50-fold decrease in Vmax/Km with TyrRS, but the mutant did not possess serine charging ability