Nucleotide insertion in the anticodon loop of a glycine transfer RNA causes missense suppression.

Abstract

We have determined the nucleotide sequences of two unusual UGG-suppressing glycine tRNAs from Escherichia coli and, as a result, have discovered a new mechanism for the generation of missense suppressors. The suppressor tRNAs translate UGG but not UGA. Each arose as a consequence of spontaneous mutational alteration of glyT, the gene for the GGA/Greading glycine tRNA of E. coli In each mutant tRNA, the change in primary structure involved the insertion ofan adenylate residue on the 3' side of the anticodon and the loss ofa modification of the uridylate residue at the 5' end of the anticodon. A "shift" of the effective anticodon by one nucleotide in the 3' direction can account for the new coding specificity of these tRNAs.(19). Nevertheless, both mutant glyT tRNAs suppressed UGG mutations occurring at the same positions in trpA (19). The conversion of a GGA/G-reading glycine tRNA to a UGG-specific suppressor alters its recognition of two positions of the codon (first and third). But suppressors of this type occur frequently, suggesting that members of this class of UGG suppressor can arise from a single mutational event. We determined the nucleotide sequence ofboth of these tRNAs and discovered a new type of missense suppressor tRNA. MATERIALS AND METHODSThe original hypothesis oftranslational suppression of missense mutations (1-3) proposed the general nature of tRNA involvement. To date, a variety of specific molecular alterations have been found to result in tRNA-mediated suppression. For example, the coding specificity of a tRNA can be altered by a change in the anticodon, either by base substitution (for review, see refs. 4-6) or by loss ofa base modification (7-9). In addition to anticodon changes, nucleotide substitutions in other parts of a tRNA molecule can result in suppression by causing attachment of an incorrect amino acid (5, 10). Moreover, a particular anticodon change was found to alter a tRNA so that it accepts a new amino acid and reads a different codon (11). An especially interesting suppressor tRNA is a mutant form of tRNA rP that can translate the termination codon UGA and the tryptophan codon, UGG (12). This mutant tRNA retains the original anticodon, and its suppressor activity is due to a single base change in the dihydrouracil arm of the molecule. How this change, distant from the anticodon, alters the codon response has not been completely resolved, but a consequent change in the tertiary structure of the molecule has been implicated (13)(14)(15). Other mutational studies (7-9, 16-19) also have indicated the importance oftRNA conformation in determining the specificity of codon response.In a recent report, we described the isolation and initial characterization of translational suppressors of a UGG missense mutation in Eschertichia coli (20). Eleven UGG suppressors were shown by genetic mapping and reversed-phase column chromatography to have alterations in glyT, the structural gene for a species of glycine tRNA. Nine suppressed both UGA and UGG mutations. This result was expe...

Structural insights into translational recoding by frameshift suppressor tRNASufJ

“…1A; Riyasaty and Atkins 1968;Riddle and Roth 1972;Yourno 1972;Prather et al 1981;Cummins et al 1982). In addition, optimal frameshift suppression resulting in higher frameshift efficiencies occurred from a four-nucleotide Watson-Crick base-pair interaction (Yarus 1982;Gaber and Culbertson 1984;Curran and Yarus 1987;Moore et al 2000;Anderson et al 2002).…”

Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit

“…1; hereafter the additional base is referred to as 33.5). Many of these suppressor tRNAs isolated in both bacteria (Riyasaty and Atkins 1968;Yourno 1972;Riddle and Carbon 1973;Prather et al 1981;Bossi and Smith 1984;Magliery et al 2001) and yeast (Cummins et al 1982;Gaber and Culbertson 1982;Sroga et al 1992) display Watson-Crick complementarity at all four anticodon positions to their quadruplet codon, suggesting that formation of 4 base pairs (bp) might cause +1 frameshifting (Roth 1981). However, +1 frameshifting still occurs, albeit at lower efficiencies, when either anticodon base 33.5 or the fourth base of the codon is singly mutated to form noncanonical base pairs, indicating that Watson-Crick pairing between these bases is not crucial (Gaber and Culbertson 1984;Curran and Yarus 1987;Weiss et al 1990).…”

Primary structure of an unusual glycine tRNA UGA suppressor

“…In contrast to the heterogeneity found at position 37 in the UGA suppressor tRNA, the A at that position in wild-type glyT tRNA is completely unmodified. This has been shown both by classical procedures of tRNA isolation and sequence analysis (12) and by more recent ones (13).…”

“…An unusual glyT UGG suppressor was found recently to employ the same anticodon (CCA) as tryptophan tRNA from wild-type E. coli but to have a completely unmodified A37 (13). These results suggest that not all tRNAs require ms2i6A for translation of codons beginning with U.…”