Rapid determination of nucleotides that define tRNA(Gly) acceptor identity.

Abstract

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 ...

Spiroplasma citri UGG and UGA tryptophan codons: sequence of the two tryptophanyl-tRNAs and organization of the corresponding genes

“…Among these six nucleotides, the modified U in the wobble position of the anticodon (U*) might be a major determinant in its nonrecognition by E. coli tryptophanyl-tRNA synthetase. This is in line with the hypothesis that in a number of tRNAs the anticodon could be involved in the recognition process (17,23).…”

Switching tRNA(Gln) identity from glutamine to tryptophan.

“…Strain YN2970 (22), mutated in release factor 2, was used for expression of DHFR suppressed by su+2UGA suppressors (20,23). DHFR was purified from 3 liters of culture from strains transformed with two compatible plasmids; containing the suppressor tRNA on a pGFIB derivative and a pACYC184 derivative, pD3op, containing the E. colifol gene with a UGA codon at position 3 (17).…”

The N-terminal Domain of Mammalian Lysyl-tRNA Synthetase Is a Functional tRNA-binding Domain

“…Nucleotide A 73 is conserved among prokaryotic tRNA Lys and, in conjunction with nucleotides U 34 , U 35 , and U 36 of the anticodon, is an important identity determinant (33,34). Therefore, the two primary interaction sites between LysRS and tRNA Lys in the E. coli system are likely to involve both the catalytic domain and the oligonucleotide-binding-folded anticodon binding domain of the synthetase (22,32).…”