The Mechanism of Action of Methionyl-tRNA Synthetase

The interactions of three methionine-specific tRNAs (initiator tRNAfMet from rabbit liver, cross-linked tRNAfMet and untreated tRNAfMet from Escherichia coli) with methionyl-tRNA synthetase from E. coli were examined. Binding parameters determined by fluorescence spectroscopy were compared with kinetic parameters derived from the aminoacylation reaction performed under comparable experimental conditions. Cross-linked tRNAfMet and untreated tRNAfMet are virtually indistinguishable on the bases of their kinetic parameters in the aminoacylation reaction, but can be differentiated by fluorescence measurements. The lower level of enzyme fluorescence quenching elicited by the cross-linked species must be related to a structural modification which has no significant repercussion on its performance in the aminoacylation reaction. The initiator tRNA from rabbit liver differs from that of E. coli in several respects. Its binding parameters, measured by fluorescence, are characterized by a two-fold higher Ka, a two-fold lower maximal level of enzyme fluorescence quenching and a less pronounced sensitivity to the addition of KCl. The lower sensitivity to KCl is also reflected in the kinetic parameters measured in the aminoacylation reaction. Several unspecific tRNAs which are not aminoacylated by the enzyme under standard conditions are able to interact with it to significant extents, as assessed by fluorescence measurements. These interactions are characterized by weak affinities and small effects on protein fluorescence, comparable in magnitude to those observed when specific tRNAs interact with the enzyme in the absence of magnesium ions.