The importance of tRNA backbone-mediated interactions with synthetase for aminoacylation

Abstract

We have identified six new aminoacylation determinants of Escherichia coli tRNAGln in a genetic and biochemical analysis of suppressor tRNA. The new determinants occupy the interior of the acceptor stem, the inside corner of the L shape, and the anticodon loop of the molecule. They supplement the primary determinants located in the anticodon and acceptor end of tRNAGln described previously. Remarkably, the three-dimensional structure of the complex between tRNAGln and glutaminyl-tRNA synthetase shows that the enzyme interacts with the phosphate-sugar backbone but not the base of every new determinant. Moreover, a small protein motif interacts with five of these determinants, and it binds proximal to the sixth. The motif also interacts with the middle base of the anticodon and with the backbones of six other nucleotides. Our results emphasize that synthetase recognition of tRNA is more elaborate than amino acid side chains of the enzyme interacting with nucleotide bases of the tRNA. Recognition also includes synthetase interaction with tRNA backbone functionalities whose distinctive locations in three-dimensional space are exquisitely determined by the tRNA sequence.

Figure 1

Cloverleaf arrangement of tRNAs corresponding to tRNA^Gln (A), the starting tRNA with arrows pointing from the bases of wild-type tRNA^Ala (UGC anticodon) that were substituted (B), mutant tRNAs with arrows pointing to mutations that improve aminoacylation (C), or mutant tRNAs with arrows pointing to mutations that improve ribosomal performance but do not alter aminoacylation (D). In A, nucleotides that determine aminoacylation of tRNA^Gln (6, 7, 9) are noted by circles for bases that are directly recognized by GlnRS and boxes for bases that facilitate the tRNA in assuming the conformation bound by the protein; thick lines show phosphate-sugar backbone-mediated interactions with GlnRS. In B, the dashed lines indicate three tertiary pairings discussed in the text. Modified nucleotides were not analyzed.

Figure 2

Activity of tRNA mutants on an X-gal indicator plate at 37°C.

Figure 3

Northern blot analysis showing aminoacyl-tRNA and uncharged tRNA of representative mutants. tRNA genes with the indicated mutations were expressed from plasmid pGFIB in XAC/A16 cells. Samples containing ≈0.05 OD₂₆₀ units of total tRNA isolated at pH 5.2 were fractionated by 6.5% PAGE in sodium acetate and urea. Percentage glutaminyl-tRNA is the average of several determinations (Table 1). We verified the glutaminyl-tRNA species by observing its signal increase from 22 to 51% when GlnRS was overproduced from plasmid pAC1.

Figure 4

Western blot analysis of GlnRS from cells expressing representative mutant tRNAs. Serial dilutions of crude extracts were fractionated by 6% SDS/PAGE. “None” means XAC/A16 cells carrying plasmid pGFIB without an amber-suppressor tRNA gene. The position of purified GlnRS marker is noted. Cells overproducing GlnRS from plasmid pAC1 showed a 4- to 8-fold increase in GlnRS signal.

Figure 5

Gel-retardation assay of glutaminyl-tRNA, EF-Tu, and GTP ternary complex. In A, binding mixtures containing 10 μM of an acid preparation of total tRNA, 10 μM EF-Tu-GTP in 46 mM Tris⋅HCl, pH 7.5, 46 mM KCl, 42 mM NH₄Cl, 6 mM MgCl₂, and 3 mM 2-mercaptoethanol were incubated 5 min at 4°C and fractionated by 6% PAGE. In B, after incubating mixtures as in A, one portion was loaded on the gel being run at 20 V and the other portion was challenged with competitor aminoacyl-tRNA (19 μM final concentration) and incubated another 10 min before loading. The competitor aminoacyl-tRNA was isolated from XAC/A16 cells carrying pGFIB without an amber- suppressor tRNA gene. EF-Tu-GDP was converted to EF-Tu-GTP just before use. Homogeneous preparations of EF-Tu were from either T. aquaticus (A, lanes 1, 2, 5, 6, 9, 10, and B) or T. thermophilus (A, lanes 3, 4, 7, 8, 11, 12). Samples were fractionated at 4°C by 6% PAGE in 25 mM Tris⋅Oac, pH 6.8, 5 mM Mg-Oac, and 5 mM NH₄-Oac, transferred by electroblot and hybridized. The gray input levels were adjusted in photoshop 4.0 from 1.00 to 2.00 in A, lanes 1–4, to better compare them with A, lanes 5–12.

Figure 6

Molecular structure of part of the tRNA^Gln–GlnRS complex. The diagram shows the Thr-316–Arg-341 motif of GlnRS and the phosphate-sugar backbone of tRNA^Gln based on the 2.5-A resolution structure of the complex (6). Amino acid side chains shown are Thr-316, Lys-317, Gln-318, Asp-319, Thr-321, Ser-326, Asn-336, and Arg-341. Spheres representing the phosphate-sugar backbone of residues that interact with the motif are shown (P of residues 5, 8, 11, 12, 13, 14, 25, 38, and 69). Blue spheres indicate nucleotides that are mutationally identified in this work; they interact with amino acid side chains indicated in blue. Arg-341 interacts with the base of U35. Table 2 lists the interacting atoms. The figure was made with insight ii (96.0.6).