Anticodon and acceptor stem nucleotides in tRNA(Gln) are major recognition elements for E. coli glutaminyl-tRNA synthetase.
The correct attachment of amino acids to their corresponding (cognate) transfer RNA catalysed by aminoacyl-tRNA synthetases is a key factor in ensuring the fidelity of protein biosynthesis. Previous studies have demonstrated that the interaction of Escherichia coli tRNA(Gln) with glutaminyl-tRNA synthetase (GlnRS) provides an excellent system to study this highly specific recognition process, also referred to as 'tRNA identity'. Accurate acylation of tRNA depends mainly on two principles: a set of nucleotides in the tRNA molecule (identity elements) responsible for proper discrimination by aminoacyl-tRNA synthetases and competition between different synthetases for tRNAs. Elements of glutamine identity are located in the anticodon and in the acceptor stem region, including the discriminator base. We report here the production of more than 20 tRNA(2Gln) mutants at positions likely to be involved in tRNA discrimination by the enzyme. Unmodified tRNA, containing the wild-type anticodon and U or G at its 5'-terminus, can be aminocylated by GlnRS with similar kinetic parameters to native tRNA(2Gln). By in vitro aminoacylation the mutant tRNAs showed decreases of up to 3 x 10(5)-fold in the specificity constant (kcat/KM)14 with the major contribution of kcat. Despite these large changes, some of these mutant tRNAs are efficient amber suppressors in vivo. Our results show that strong elements for glutamine identity reside in the anticodon region and in positions 2 and 3 of the acceptor stem, and that the contribution of different identity elements to the overall discrimination varies significantly. We discuss our data in the light of the crystal structure of the GlnRS:tRNA(Gln) complex.
Jahn
M
M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Rogers
M J
MJ
Söll
D
D
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000926:Anticodon / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012351:RNA, Transfer, Gln / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000926:Anticodon; D012351:RNA, Transfer, Gln; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase