Structural basis for misaminoacylation by mutant E. coli glutaminyl-tRNA synthetase enzymes.
A single-site mutant of Escherichia coli glutaminyl-synthetase (D235N, GlnRS7) that incorrectly acylates in vivo the amber suppressor supF tyrosine transfer RNA (tRNA(Tyr] with glutamine has been described. Two additional mutant forms of the enzyme showing this misacylation property have now been isolated in vivo (D235G, GlnRS10; I129T, GlnRS15). All three mischarging mutant enzymes still retain a certain degree of tRNA specificity; in vivo they acylate supE glutaminyl tRNA (tRNA(Gln] and supF tRNA(Tyr) but not a number of other suppressor tRNA's. These genetic experiments define two positions in GlnRS where amino acid substitution results in a relaxed specificity of tRNA discrimination. The crystal structure of the GlnRS:tRNA(Gln) complex provides a structural basis for interpreting these data. In the wild-type enzyme Asp235 makes sequence-specific hydrogen bonds through its side chain carboxylate group with base pair G3.C70 in the minor groove of the acceptor stem of the tRNA. This observation implicates base pair 3.70 as one of the identity determinants of tRNA(Gln). Isoleucine 129 is positioned adjacent to the phosphate of nucleotide C74, which forms part of a hairpin structure adopted by the acceptor end of the complexed tRNA molecule. These results identify specific areas in the structure of the complex that are critical to accurate tRNA discrimination by GlnRS.
Perona
J J
JJ
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Swanson
R N
RN
Rould
M A
MA
Steitz
T A
TA
Söll
D
D
D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid; D001665:Binding Sites; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005973:Glutamine / Q000378:metabolism; D006860:Hydrogen Bonding; D007532:Isoleucine; D015394:Molecular Structure; D009154:Mutation; D012351:RNA, Transfer, Gln / Q000378:metabolism; D012365:RNA, Transfer, Tyr; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013489:Suppression, Genetic
D012351:RNA, Transfer, Gln; D012365:RNA, Transfer, Tyr; D007532:Isoleucine; D005973:Glutamine; D001224:Aspartic Acid; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase