JBC, Vol. 252, Issue 3, 814-819, Feb, 1977
Alteration of the kinetic parameters for aminoacylation of Escherichia coli formylmethionine transfer RNA by modification of an anticodon base
L. H. Schulman and H. Pelka
Treatment of Escherichia coli formylmethionine tRNA with 2 M sodium
bisulfite, pH 7.0, in 10 mM MgCl2 at 25 degrees results in formation of
uridine/bisulfite adducts at U18 in the dihydrouridine loop, U37 in the
anticodon, and U48 in the variable loop. Two products, corresponding to the
two diastereoisomers of 5,6-dihydrouridine-6-sulfonate, are formed at each
reactive site in the tRNA. Although none of the modifications cause
complete loss of methionine acceptor activity, the modified tRNA is
amino-acylated at a reduced rate and has a decreased affinity for E. coli
methionyl-tRNA synthetase. Aminoacylation of [35S]bisulfite-labeled
tRNAfMet with a limiting amount of purified enzyme followed by separation
of the acylated and unacylated molecules and structural analysis has shown
that the presence of a specific diastereoisomer of the uridine/bisulfite
adduct in the anticodon base U37 alters the kinetic parameters for
aminoacylation of tRNAfMet.
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