Although the genetic code for protein was established in the 1960's, the basis for amino acid identity of transfer RNA (tRNA) has remained unknown. To investigate the identity of a tRNA, the nucleotides at three computer-identified positions in tRNAPhe (phenylalanine tRNA) were replaced with the corresponding nucleotides from tRNAAla (alanine tRNA). The identity of the resulting tRNA, when examined as an amber suppressor in Escherichia coli, was that of tRNAAla.

The specificity of tRNA(Arg) (arginine transfer RNA) for aminoacylation (its acceptor identity) were first identified by computer analysis and then examined with amber suppressor tRNAs in Escherichia coli. On replacing two nucleotides in tRNA(Phe) (phenylalanine transfer RNA) with the corresponding nucleotides from tRNA(Arg), the acceptor identity of the resulting tRNA was changed to that of tRNA(Arg). The nucleotides used in the identity transformation occupy a "variable pocket" structure on the surface of the tRNA molecule where two single-stranded loop segments interact. The middle nucleotide in the anticodon also probably contributes to the interaction, since an amber suppressor of tRNA(Arg) had an acceptor identity for lysine as well as arginine.

We have constructed an opal suppressor system in Escherichia coli to complement an existing amber suppressor system to study the structural basis of tRNA acceptor identity, particularly the role of middle anticodon nucleotide at position 35. The opal suppressor tRNA contains a UCA anticodon and the mRNA of the suppressed protein (which is easily purified and sequenced) contains a UGA nonsense triplet. Opal suppressor tRNAs of two tRNAAg isoacceptor sequences each gave arginine in the suppressed protein, while the corresponding amber suppressors with U35 in their CUA anticodons each gave arginine plus a second amino acid in the suppressed protein. Since C35 but not U35 is present in the anticodon of wild-type tRNAAg molecules, while the first anticodon position contains either C34 or U34, these results establish that C35 contributes to tRNAArg acceptor identity. Initial characterizations of opal suppressor tRNAArg mutants by suppression efficiency measurements suggest that the fourth nucleotide from the 3' end of tRNAArg (A73 or G73 in different isoacceptors) also contributes to tRNAArg acceptor identity. Wild-type and mutant versions of opal and amber tRNALYS suppressors were examined, revealing that U35 and A73 are important determinants of tRNALyS acceptor identity. The aminoacylation specificity of tRNA ("tRNA acceptor identity") is essential for protein synthesis. The structural features in tRNA that determine tRNA acceptor identity have been studied in several ways: by measuring in vitro aminoacylation of tRNAs that differ structurally from corresponding wild-type tRNAs (1-16); by determining the interacting surfaces in complexes of tRNAs and aminoacyl-tRNA synthetases (17-23); by comparing tRNA sequences (24-27); and by determining the in vivo amino acid specificities of suppressor tRNAs (28-44). The acceptor identity of tRNA results from the tRNA's productive interaction with the cognate aminoacyl-tRNA synthetase and nonproductive interactions with all other aminoacyl-tRNA synthetase enzymes. The net outcome of both types of interactions is obtained with a suppressor tRNA. For analysis, a suppressor tRNA gene present in a plasmid is inserted into a cell, and, because of its distinctive codon recognition properties, the acceptor identity of the transcribed suppressor tRNA is mirrored by the amino acid recovered in a suppressed protein. Mutants of a suppressor tRNA can pinpoint the specific nucleotides that determine tRNA acceptor identity when the amino acid recovered in the suppressed protein is altered.We previously reported (38) that the acceptor identity of Escherichia coli tRNAAr9 is partially determined by the adenosine residue at position 20 (A20) in the variable pocket (38). Our work was based on a computer analysis of tRNA sequences and subsequent sequencing of suppressed protein produced by mutants of amber suppressor tRNAs. We also suggested that the cytidine residue at position 35 (C35) in the wild-type anticodon contributes to tRNAAr9 acceptor identity because amber suppressor (U35) tR...

The aminoacylation specificity ("acceptor identity") of transfer RNAs (tRNAs) has previously been associated with the position of particular nucleotides, as opposed to distinctive elements of three-dimensional structure. The contribution of a G.U wobble pair in the acceptor helix of tRNA(Ala) to acceptor identity was examined with synthetic amber suppressor tRNAs in Escherichia coli. The acceptor identity was not affected by replacing the G.U wobble pair in tRNA(Ala) with a G.A, C.A, or U.U wobble pair. Furthermore, a tRNA(Ala) acceptor identity was conferred on tRNA(Lys) when the same site in the acceptor helix was replaced with any of several wobble pairs. Additional data with tRNA(Ala) show that a substantial acceptor identity was retained when the G.U wobble pair was translocated to another site in the acceptor helix. These results suggest that the G.U wobble pair induces an irregularity in the acceptor helix of tRNA(Ala) to match a complementary structure in the aminoacylating enzyme.

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