Recognition of bases in Escherichia coli tRNA(Gln) by glutaminyl-tRNA synthetase: a complete identity set.
The fidelity of protein biosynthesis rests largely on the correct aminoacylation of transfer RNAs by their cognate aminoacyl-tRNA synthetases. Previous studies have demonstrated that the interaction of Escherichia coli tRNA(Gln) with glutaminyl-tRNA synthetase (GlnRS) provides an excellent system for studying the basis of this highly specific recognition process. Correct aminoacylation depends on the set of nucleotides (identity elements) in tRNA(Gln) responsible for correct interaction with GlnRS. Specific contacts between tRNA(Gln) and GlnRS include the 2-amino group of guanosines. Therefore, we made a set of tRNA(Gln) variants in which specific guanosines were replaced by inosine using recombinant RNA technology. This resulted in a set of tRNAs that varied by single deletions of the amino group from guanine residues, thus allowing us to test the functional importance of these contacts. In addition, a number of mutants were made by transcription of mutated tRNA genes with base changes at position 10, 16 or 25. In vitro aminoacylation of these mutants showed decreases in the specificity constant (kcat/KM) of up to 300-fold, with kcat being the parameter most affected. These experiments reveal G10 as a new element of glutamine identity. In addition, the interaction of G2, G3 and G10 with GlnRS via the 2-amino group is significant for tRNA discrimination. Based on these results, and on earlier data, we propose a complete set of bases as identity elements for tRNA(Gln).
Hayase
Y
Y
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Jahn
M
M
Rogers
M J
MJ
Sylvers
L A
LA
Koizumi
M
M
Inoue
H
H
Ohtsuka
E
E
Söll
D
D
D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012351:RNA, Transfer, Gln / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D012351:RNA, Transfer, Gln; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase