Alteration of the kinetic parameters for aminoacylation of Escherichia coli formylmethionine transfer RNA by modification of an anticodon base.
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.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000926:Anticodon / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D009239:N-Formylmethionine; D012343:RNA, Transfer / Q000378:metabolism*; D013447:Sulfites
D000926:Anticodon; D013447:Sulfites; D009239:N-Formylmethionine; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase