The structural features of the G.U wobble pair in Escherichia coli alanine transfer RNA (tRNA(Ala)) that are associated with aminoacylation by alanyl-tRNA synthetase (AlaRS) were investigated in vivo for wild-type tRNA(Ala) and mutant tRNAs with G.U substitutions. tRNA(Ala) with G.U, C.A, or G.A gave similar amounts of charged tRNA(Ala) and supported viability of E. coli lacking chromosomal tRNA(Ala) genes. tRNA(Ala) with G.C was inactive. Recognition of G.U by AlaRS thus requires more than the functional groups on G.U in a regular helix and may involve detection of a helical distortion.

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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Expression of the genetic code depends on the recognition ofspecific tRNAs by the enzymes that aminacylate them. A computer comparison oftRNA sequences coupled with analysis of mutant nonsense-suppressor tRNAs has revealed the structural features that distinguish the acceptor identity of Escherichia coli tRNAGIY from tRNAs that accept phenylalanine, arginine, lysine, and glutamine. On replacement of several nucleotides in the acceptor stem and anticodon of the latter tRNAs with tRNAGlybderived residues, the resulting molecules acquired a tRNAGIy identity.Genes specify the structures of proteins that are made in cells. Following transcription of a gene's sequence into mRNA, 20 types of tRNA molecules translate the codons into the amino acid sequence of the protein. Each acceptor type oftRNA has two specificities, one for the codon and the other for the amino acid. The specificity for the amino acid, termed tRNA acceptor identity, is dictated by some distinctive molecular structure in the tRNA that is manifested during its aminoacylation by the cognate aminoacyl-tRNA synthetase enzyme. The acceptor identity of a tRNA is the net result of two types of interactions, the productive interaction of the tRNA with the cognate aminoacyl-tRNA synthetase and the nonproductive interactions with all other aminoacyl-tRNA synthetases.The structural basis of tRNA acceptor identity is being elucidated with mutant tRNAs that have acquired new identities following specific changes in a few nucleotides in their sequences. The tRNA mutants are relatively simple to construct with synthetic DNAs, and in some cases their sequences are designed in conjunction with computer analysis of tRNA sequences. Seven of 20 possible tRNA acceptor types have been characterized thus far, including tRNAs that accept serine (1, 2), methionine (3), alanine (4, 5), phenylalanine (6, 7), arginine (8, 9), glutamine (10), and lysine (11). Here we present a description of the determinants of Escherichia coli tRNA61Y. Sequence Comparisons and Genetic AnalysesWe aligned 67 E. coli tRNA sequences (12) according to their common cloverleaf secondary structure so that the nucleotide residues at corresponding positions could be compared. A computer program (13) then identified the nucleotides that were conserved in the 5 tRNAGly isoacceptor sequences while differing from the nucleotides in the 62 other tRNA acceptor types. This revealed that no nucleotide at a single position distinguishes tRNAGly from all others. However, 15 of 176,700 possible two-nucleotide combinations (the combinations of 76 nucleotides taken 2 at a time from a tRNAGlY composite sequence compared with each of 62 other tRNA sequences) were distinguishing characteristics of tRNAGlY (Fig. 1). Although the uridine at position 73 (U73) was most prevalent and thus the best structural predictor, it must function with other nucleotides to discriminate tRNAGly. Fig. 2 shows the sequences ofthree tRNAGly isoacceptors and the location of residue U73 on the acceptor stem of the molecule. We show ...