Methionyl-tRNA synthetase-induced conformational change of Escherichia coli tRNAfMet

Ribonuclease T2, nuclease S1, and snake venom phosphodiesterase were used as a structural probe for investigation of the interaction between Escherichia coli tRNAfMet and methionyl-tRNA synthetase, and the cleavage sites were analyzed by a rapid sequencing gel electrophoresis of 5'-32P-labeled tRNA. Both endonucleases cleaved the D-loop of synthetase-bound tRNA much more extensively than that of the free tRNA. Positions of A14, G15, A22, and G23 in the D-loop and C35 in the anticodon of the synthetase-bound tRNA were more susceptible to RNase T2. The synthetase-bound tRNA was predominantly cleaved by nuclease S1 at position of G15, G19, G20, and G23 in the D-loop and G2 in the acceptor stem. In contrast, the synthetase-bound tRNA was more resistant to the 3'-exonuclease, snake venom phosphodiesterase, than was the free tRNA molecule. These results suggest conformational change of the tRNA by the synthetase binding which weakened tertiary interaction between the D-loop and T psi C-loop/extra-loop. Production of acid-soluble radioactivity was also examined in the limited digestion of 5'-32P-labeled tRNA or 3'-14C-labeled methionyl-tRNA. The synthetase enhanced the release of acid-soluble oligonucleotides from the 5'-end of the tRNA but suppressed that from the 3'-end of the molecule. These results are consistent with that obtained by gel electrophoresis.     

Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. coli tRNAfMet by E. coli Met-tRNA synthetase

Derivatives of E. coli tRNAfMet containing single base substitutions at the wobble position of the anticodon have been enzymatically synthesized in vitro. The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMet with RNase A. RNA ligase is then used to join each of four trinucleotides, NAU, to the 5' half molecule and to subsequently link the 3' and modified 5' fragments to regenerate the anticodon loop. Synthesis of intact tRNAfMet containing the anticodon CAU by this procedure yields a product which is indistinguishable from native tRNAfMet with respect to its ability to be aminoacylated by E. coli methionyl-tRNA synthetase. Substitution of any other nucleotide at the wobble position of tRNAfMet drastically impairs the ability of the synthetase to recognize the tRNA. Measurement of methionine acceptance in the presence of high concentrations of pure enzyme has established that the rate of aminoacylation of the AAU, GAU and UAU anticodon derivatives of tRNAfMet is four to five orders of magnitude slower than that of the native or synthesized tRNA containing C as the wobble base. In addition, the inactive tRNA derivatives fail to inhibit aminoacylation of normal tRNAfMet, indicating that they bind poorly to the enzyme. These results support a model involving direct interaction between Met-tRNA synthetase and the C in the wobble position during aminoacylation of tRNAfMet.     

Tertiary structure of Escherichia coli tRNA(3Thr) in solution and interaction of this tRNA with the cognate threonyl-tRNA synthetase

The solution structure of Escherichia coli tRNA(3Thr) (anticodon GGU) and the residues of this tRNA in contact with the alpha 2 dimeric threonyl-tRNA synthetase were studied by chemical and enzymatic footprinting experiments. Alkylation of phosphodiester bonds by ethylnitrosourea and of N-7 positions in guanosines and N-3 positions in cytidines by dimethyl sulphate as well as carbethoxylation of N-7 positions in adenosines by diethyl pyrocarbonate were conducted on different conformers of tRNA(3Thr). The enzymatic structural probes were nuclease S1 and the cobra venom ribonuclease. Results will be compared to those of three other tRNAs, tRNA(Asp), tRNA(Phe) and tRNA(Trp), already mapped with these probes. The reactivity of phosphates towards ethylnitrosourea of the unfolded tRNA was compared to that of the native molecule. The alkylation pattern of tRNA(3Thr) shows some similarities to that of yeast tRNA(Phe) and mammalian tRNA(Trp), especially in the D-arm (positions 19 and 24) and with tRNA(Trp), at position 50, the junction between the variable region and the T-stem. In the T-loop, tRNA(3Thr), similarly to the three other tRNAs, shows protections against alkylation at phosphates 59 and 60. However, tRNA(3Thr) is unique as far as very strong protections are also found for phosphates 55 to 58 in the T-loop. Compared with yeast tRNA(Asp), the main differences in reactivity concern phosphates 19, 24 and 50. Mapping of bases with dimethyl sulphate and diethyl pyrocarbonate reveal conformational similarities with yeast tRNA(Phe). A striking conformational feature of tRNA(3Thr) is found in the 3'-side of its anticodon stem, where G40, surrounded by two G residues, is alkylated under native conditions, in contrast to other G residues in stem regions of tRNAs which are unreactive when sandwiched between two purines. This data is indicative of a perturbed helical conformation in the anticodon stem at the level of the 30-40 base pairs. Footprinting experiments, with chemical and enzymatic probes, on the tRNA complexed with its cognate threonyl-tRNA synthetase indicate significant protections in the anticodon stem and loop region, in the extra-loop, and in the amino acid accepting region. The involvement of the anticodon of tRNA(3Thr) in the recognition process with threonyl-tRNA synthetase was demonstrated by nuclease S1 mapping and by the protection of G34 and G35 against alkylation by dimethyl sulphate. These data are discussed in the light of the tRNA/synthetase recognition problem and of the structural and functional properties of the tRNA-like structure present in the operator region of the thrS mRNA.     

Modification of specific lysine residues in E. coli methionyl-tRNA synthetase by crosslinking to E. coli formylmethionine tRNA

A protein affinity labeling derivative of E. coli tRNAfMet has been prepared which carries an average of one reactive side chain per molecule, distributed over four structural regions. Each side chain contains a disulfide bond capable of reaction with cysteine residues and an N-hydroxysuccinimide ester group capable of coupling to lysine epsilon-amino groups in proteins. Reaction of the modified tRNA with E. coli methionyl-tRNA synthetase leads to crosslinking only by reaction with lysine residues in the protein. Examination of the tRNA present in the crosslinked complex reveals that the enzyme is coupled to side chains attached to the 5' terminal nucleotide, the dihydrouridine loop, the anticodon and the CCA sequence. Digestion of the crosslinked enzyme with trypsin followed by peptide mapping reveals that the major crosslinking reactions occur at four specific lysine residues, with minor reaction at two additional sites. Native methionyl-tRNA synthetase contains 90 lysine residues, 45 in unique sequences of the dimeric alpha 2 enzyme. Crosslinking of the protein to different regions in tRNAfMet thus occurs with the high degree of selectivity necessary for use in determining the peptide sequences which are near specific nucleotide sequences of tRNA bound to the protein.     

Binding of the anticodon domain of tRNA(fMet) to Escherichia coli methionyl-tRNA synthetase

A stem and loop RNA domain carrying the methionine anticodon (CAU) was designed from the tRNA(fMet) sequence and produced in vitro. This domain makes a complex with methionyl-tRNA synthetase (Kd = 38(+/- 5) microM; 25 degrees C, pH 7.6, 7 mM-MgCl2). The formation of this complex is dependent on the presence of the cognate CAU anticodon sequence. Recognition of this RNA domain is abolished by a methionyl-tRNA synthetase mutation known to alter the binding of tRNA(Met).     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

In vitro conversion of a methionine to a glutamine-acceptor tRNA

A derivative of Escherichia coli tRNAfMet containing an altered anticodon sequence, CUA, has been enzymatically synthesized in vitro. The variant tRNA was prepared by excision of the normal anticodon, CAU, in a limited digestion of intact tRNAfMet with RNase A, followed by insertion of the CUA sequence into the anticodon loop with T4 RNA ligase and polynucleotide kinase. The altered methionine tRNA showed a large enhancement in the rate of aminoacylation by glutaminyl-tRNA synthetase and a large decrease in the rate of aminoacylation by methionyl-tRNA synthetase. Measurement of kinetic parameters for the charging reaction by the cognate and noncognate enzymes revealed that the modified tRNA is a better acceptor for glutamine than for methionine. The rate of mischarging is similar to that previously reported for a tryptophan amber suppressor tRNA containing the anticodon CUA, su+7 tRNATrp, which is aminoacylated with glutamine both in vivo and in vitro [Yaniv, M., Folk, W. R., Berg, P., & Soll, L. (1974) J. Mol. Biol. 86, 245-260; Yarus, M., Knowlton, R. E., & Soll, L. (1977) in Nucleic Acid-Protein Recognition (Vogel, H., Ed.) pp 391-408, Academic Press, New York]. The present results provide additional evidence that the specificity of aminoacylation by glutaminyl-tRNA synthetase is sensitive to small changes in the nucleotide sequence of noncognate tRNAs and that uridine in the middle position of the anticodon is involved in the recognition of tRNA substrates by this enzyme.     

Recognition of tRNAs by aminoacyl-tRNA synthetases: Escherichia coli tRNAMet and E. coli methionyl-tRNA synthetase

In previous work we identified several specific sites in Escherichia coli tRNAfMet that are essential for recognition of this tRNA by E. coli methionyl-tRNA synthetase (MetRS) (EC 6.1.1.10). Particularly strong evidence indicated a role for the nucleotide base at the wobble position of the anticodon in the discrimination process. We have now investigated the aminoacylation activity of a series of tRNAfMet derivatives containing single base changes in each position of the anticodon. In addition, derivatives containing permuted sequences and larger and smaller anticodon loops have been prepared. The variant tRNAs have been enzymatically synthesized in vitro by using T4 RNA ligase (EC 6.5.1.3). Base substitutions in the wobble position have been found to reduce aminoacylation rates by at least five orders of magnitude. Derivatives having base substitutions in the other two positions of the anticodon are aminoacylated 55-18,500 times slower than normal. Nucleotides that have specific functional groups in common with the normal anticodon bases are better tolerated at each of these positions than those that do not. A tRNAfMet variant having a six-membered loop containing only the CA sequence of the anticodon is aminoacylated still more slowly, and a derivative containing a five-membered loop is not measurably active. The normal loop size can be increased by one nucleotide with a relatively small effect on the rate of aminoacylation, which indicates that the spatial arrangement of the nucleotides is less critical than their chemical nature. We conclude from these data that recognition of tRNAfMet requires highly specific interactions of MetRS with functional groups on the nucleotide bases of the anticodon sequence. Several other aminoacyl-tRNA synthetases are known to require one or more anticodon bases for efficient aminoacylation of their tRNA substrates, and data from other laboratories suggest that anticodon sequences may be important for accurate discrimination between cognate and noncoagnate tRNAs by these enzymes.     

Replacement and insertion of nucleotides at the anticodon loop of E. coli tRNAMetf by ligation of chemically synthesized ribooligonucleotides.

Insertion of the four major nucleotides at the 5'-side of the anticodon triplet of E. coli tRNAMetf was performed by joining of the half molecules obtained by limited digestion with RNase A and the chemically synthesized tetranucleotide pN-C-A-U using RNA ligase. Insertion of U-U at the 5'-side or A and A-A at the 3'-side of the anticodon were also performed using U-U-C-A-U, C-A-U-A and C-A-U-A-A. The constant U next to the 5'-side of the anticodon was replaced with A and C by ligation of A-C-A-U and C-C-A-U to the 5'-half molecule which had been treated with periodate plus lysine, followed by joining to the 3'-half. These modified tRNAs were tested for their ability to accept methionine with the methionyl-tRNA synthetase of E. coli. The affinity of these analogs for the synthetase decreased more extensively when the insertion was at the 3'-side of the anticodon triplet. Insertion of mononucleotides at the 5'-side or replacement of the constant U next to the 5'-side of the anticodon did not affect aminoacylation drastically. This may mean that the 3'-side of the anticodon loop of tRNA is one of the major recognition sites for the methionyl-tRNA synthetase.     

Topographic modeling of free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer: bending of the 3'-terminal arm in tRNAfMet

Conformations of tRNAfMet, free and methionyl-tRNA synthetase bound forms, are analyzed by using singlet-singlet energy transfer as a spectroscopic ruler. tRNAfMet(8-13,3'-Flc), tRNAfMet(8-13,D-Etd), and tRNAfMet(3'-Flc,D-Etd) are prepared by sequential chemical modifications. The methionyl-tRNA synthetase binding affinity of these double-labeled tRNAfMets is similar to those of unmodified tRNAfMet. The fluorescence properties of the individual fluorophore in these tRNAs, including emission spectra, anisotropy, and quenching by methionyl-tRNA synthetase, are similar to those of single-labeled tRNAfMet. The transfer efficiencies of double-labeled tRNAfMets, as determined by both donor quenching and sensitized emission, showed efficient energy transfer in all cases. Random orientation being assumed, the apparent distances are 25 A between 8-13 and D20, 44 A between 8-13 and the 3'-terminus, and 49 A between the 3'-terminus and D20, respectively, in free tRNAfMet. Upon binding of methionyl-tRNA synthetase, the apparent distances are 25 A between 8-13 and D20, 45 A between 8-13 and the 3'-terminus, and 54 A between the 3'-terminus and D20, respectively. These results provide topographic models of these specific locations in free and methionyl-tRNA synthetase bound tRNAfMet and suggest that the immobilized 3'-terminal arm in the amino acid acceptor stem bends toward the inner loop of the L-shaped tRNA upon binding of methionyl-tRNA synthetase.     

Alteration of the kinetic parameters for aminoacylation of Escherichia coli formylmethionine transfer RNA by modification of an anticodon base.

Treatment of Escherichia coli formylmethionine tRNA with 2 M sodium bisulfite, pH 7.0, in 10 mM MgCl2 at 25 degrees results in formation of uridine/bisulfite adducts at U18 in the dihydrouridine loop, U37 in the anticodon, and U48 in the variable loop. Two products, corresponding to the two diastereoisomers of 5,6-dihydrouridine-6-sulfonate, are formed at each reactive site in the tRNA. Although none of the modifications cause complete loss of methionine acceptor activity, the modified tRNA is amino-acylated at a reduced rate and has a decreased affinity for E. coli methionyl-tRNA synthetase. Aminoacylation of [35S]bisulfite-labeled tRNAfMet with a limiting amount of purified enzyme followed by separation of the acylated and unacylated molecules and structural analysis has shown that the presence of a specific diastereoisomer of the uridine/bisulfite adduct in the anticodon base U37 alters the kinetic parameters for aminoacylation of tRNAfMet.     

Tertiary structure of animal tRNATrp in solution and interaction of tRNATrp with tryptophanyl-tRNA synthetase

Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N-7 in guanosines and N-3 in cytidines by dimethyl sulfate and carbethoxylation of N-7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNATrp in solution as well as its interactions with tryptophanyl-tRNA synthetase. The phosphate reactivity towards ethylnitrosourea of unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNATrp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and T psi arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNAPhe since beef tRNATrp showed a more stable D stem and a less stable T psi stem. When alkylation by ethylnitrosourea was studied with the tRNATrp X tryptophanyl-tRNA synthetase complex we found that phosphates located at the 5' side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N-3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase X tRNA complex as compared with the modification in free tRNATrp; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNATrp in the recognition process with tryptophanyl-tRNA synthetase was confirmed in nuclease S1 mapping experiments.     

tRNA recognition site of Escherichia coli methionyl-tRNA synthetase

We have previously shown that anticodon bases are essential for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase (MetRS) [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759] and that the enzyme tightly binds to C34 at the wobble position of E. coli initiator methionine tRNA (tRNAfMet) [Pelka, H., & Schulman, L. H. (1986) Biochemistry 25, 4450-4456]. We have also previously demonstrated that an affinity labeling derivative of tRNAfMet can be quantitatively cross-linked to the tRNA binding site of MetRS [Valenzuela, D., & Schulman, L. H. (1986) Biochemistry 25, 4555-4561]. Here, we have determined the site in MetRS which is cross-linked to the anticodon of tRNAfMet, as well as the location of four additional cross-links. Only a single peptide, containing Lys465, is covalently coupled to C34, indicating that the recognition site for the anticodon is close to this sequence in the three-dimensional structure of MetRS. The D loop at one corner of the tRNA molecule is cross-linked to three peptides, containing Lys402, Lys439, and Lys596. The 5' terminus of the tRNA is cross-linked to Lys640, near the carboxy terminus of the enzyme. Since the 3' end of tRNAfMet is positioned close to the active site in the N-terminal domain [Hountondji, C., Blanquet, S., & Lederer, F. (1985) Biochemistry 24, 1175-1180], this result indicates that the carboxy ends of the two polypeptide chains of native dimeric MetRS are folded back toward the N-terminal domain of each subunit.     

The identification of the sites of tRNA(Ser)(GCU) interaction in bovine liver with homologous aminoacyl-tRNA-synthetase by the chemical modification method]

Interaction of the bovine liver tRNA(GCUSer) having a long variable loop, with the cognate aminoacyl-tRNA synthetase has been studied by alkylation with ethylnitrosourea. It was shown that seryl-tRNA synthetase protects 3'-phosphates of nucleotides 12, 13 in D-stem and 45-47-, 47 G.-, 47 H-variable stem of tRNA(GCUreS) from alkylation. An anticodon loop of tRNA(GCUSer) did not interact with seryl-tRNA synthetase.     

tRNAfMet-induced conformational transition at the intersubunit domain of fluorescent-labeled methionyl-tRNA synthetase

Conformational transition in methionyl-tRNA synthetase upon binding of tRNAfMet, whose binding shows strong negative cooporativity, was analyzed by fluorescence spectroscopy. The fluorescent probe N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS) reacts with native methionyl-tRNA synthetase in a nearly stoichiometric amount (2 per dimer) without affecting enzyme activity. The probe is shown by controlled trypsinization to be located in a 130 amino acid fragment at the C-terminus joining the subunits. The emission and excitation spectra, rotational freedom, and solvent accessibility of the fluorophore in AEDANS-methionyl-tRNA synthetase are analyzed. The results suggest that the probe is localized in a nonpolar environment, nearly immobile relative to methionyl-tRNA synthetase yet fully accessible to the solvent. Upon binding of tRNAfMet, the fluorescence intensity in AEDANS-methionyl-tRNA synthetase was appreciably reduced without a shift in the emission or excitation spectra. Lifetime measurement shows that a static mechanism accounts for the observed quenching. Furthermore, the remaining emitting AEDANS becomes effectively shielded from solvent molecules. These results suggest an unsymmetric conformational transition at the intersubunit domains of the two subunits in methionyl-tRNA synthetase upon binding one molecule of tRNAfMet.     

Comparison of Escherichia coli tRNAPhe in the free state, in the ternary complex and in the ribosomal A and P sites by chemical probing

tRNAPheE.coli was modified at accessible guanosine, cytidine, and adenosine residues using the chemical modification method described by Peattie and Gilbert [Proc. Natl Acad. Sci. USA, 77, 4679-4689 (1980)]. Modification characteristics of the tRNA in the free state, in the ternary complex with elongation factor EF-Tu and GTP and in the ribosomal A and P sites were compared. A special procedure was devised to monitor, exclusively, tRNA molecules in the aminoacylated state. In the free tRNA, the most reactive bases are confined to the A73-C-C-A sequence of the aminoacyl stem, the anticodon loop, the D-loop and the extra loop and the results correlate well with the three-dimensional structure of tRNAPheyeast determined by X-ray studies. The pattern of reactivity was not affected either by charging the tRNA with phenylalanine or by labelling the 3' terminus with pCp. In the ternary complex, with elongation factor EF-Tu and GTP, changes in modification were observed at two sites, A73-C-C-A at the 3' terminus and C-13 and C-17 in the D-loop region, which are about 6 nm apart; no difference was observed in the anticodon loop. tRNAPhe bound at the ribosomal A or P sites exhibited similar, but not identical, modification patterns. Whereas nucleotides C-74 and C-75 were strongly protected at both sites, the adjacent A-73 showed an enhanced reactivity in the A site. The anticodon region G34-A-A-ms2.6(1)A was also strongly protected at both sites. In addition, nucleotide A-21 was protected during A-site, but not P-site, binding.     

Methionyl-tRNA synthetase induced 3'-terminal and delocalized conformational transition in tRNAfMet: steady-state fluorescence of tRNA with a single fluorophore

Five species of tRNAfMet labeled with a single fluorophore are prepared to analyze the conformational changes at the 3'-end, at dihydrouridine, and at thiouridine in tRNAfMet upon binding of methionyl-tRNA synthetase. The emission and excitation spectra, anisotropy, and solvent accessibility of the fluorophore in each of the modified tRNAfMet's are determined in the absence and presence of methionyl-tRNA synthetase. The results are consistent with the following. The probes at the 3'-end are in a nonpolar environment, mobile relative to the tRNA molecule, and fully exposed to the solvent. The probes at dihydrouridine are partially stacked over the neighboring bases, nearly immobile, and relatively inaccessible. The S8-C13 cross-linked product is rigid. Upon binding of methionyl-tRNA synthetase, the probes at the 3'-terminus become localized in a less polar environment, highly immobilized, and effectively shielded against solvent access, while the probes at dihydrouridine appear to be partially unstacked from the neighboring base and become slightly more accessible for solvent. Singlet-singlet energy transfer between the intrinsic protein fluorescence and the fluorophores in modified tRNA's was observed by sensitized emission for tRNAfMet modified at the 3'-end and for S8-C13 but not for tRNAfMet's modified at dihydrouridine. These results suggest that dihydrouridine in tRNAfMet is oriented away from methionyl-tRNA synthetase in the tRNA-enzyme complex.     

Structural insights into translational recoding by frameshift suppressor tRNASufJ

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5′ or 3′ direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNA^SufJ, a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNA^SufJ contains an insertion 5′ to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASL^SufJ or tRNA^SufJ does not affect its affinity for the A site of the ribosome. Structural analyses of both ASL^SufJ and ASL^Thr bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34–37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASL^SufJ imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNA^SufJ during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting.