Mj. Rogers et al., FUNCTIONAL COMMUNICATION IN THE RECOGNITION OF TRANSFER-RNA BY ESCHERICHIA-COLI GLUTAMINYL-TRANSFER RNA-SYNTHETASE, Proceedings of the National Academy of Sciences of the United Statesof America, 91(1), 1994, pp. 291-295
Wild-type Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1
.18) poorly aminoacylates opal suppressors (GLN) derived from tRNA(Gln
). Mutations in glnS (the gene encoding GlnRS) that compensate for imp
aired aminoacylation were isolated by genetic selection. Two glnS muta
nts were obtained by using opal suppressors differing in the nucleotid
es composing the base pair at 3.70: glnS113 with an Asp-235 --> Asn ch
ange selected with GLNA3U70 (GLN carrying G3 --> A and C70 --> U chang
es), and glnS114 with a Gln-318 --> Arg change selected with GLNU70 (G
LN carrying a C70 --> U change). The Asp-235 --> Asn change was identi
fied previously by genetic selection. Additional mutants were isolated
by site-directed mutagenesis followed by genetic selection; the mutan
t enzymes have single amino acid changes (Lys-317 --> Arg and Gln-318
--> Lys). A number of mutants with no phenotype also were obtained ran
domly. In vitro aminoacylation of a tRNA(Gln) transcript by GlnRS enzy
mes with Lys-317 --> Arg, Gln-318 --> Lys, or Gln-318 --> Arg changes
shows that the enzyme's kinetic parameters are not greatly affected by
the mutations. However, aminoacylation of a tRNA(Gln) transcript with
an opal (UCA) anticodon shows that the specificity constants (k(cat)/
K(m)) for the mutant enzymes were 5-10 times above that of the wild-ty
pe GlnRS. Interactions between Lys-317 and Gln-318 with the inside of
the L-shaped tRNA and with the side chain of Gln-234 provide a connect
ion between the acceptor end-binding and anticodon-binding domains of
GlnRS. The GlnRS mutants isolated suggest that perturbation of the int
eractions with the inside of the tRNA L shape results in relaxed antic
odon recognition.