Transfer RNA Labeling of Escherichia coli Methionyl-tRNA Transformylase

Abstract

Homogeneous methionyl-tRNA transformylase from Escherichia coli can react with periodate-treated tRNAMetf to form a Schiff's base through a free amino group (probably the epsilon-amino group of a lysine) and the 2',3'-aldehyde groups created at the 3'-terminal ribose of tRNA. This reaction is reflected by the loss of activity of the enzyme at saturating tRNA dialdehyde and upon reduction of the Schiff's base by NaBH4. the kinetics of inactivation (37 degrees C, pH 8.5) level off at 50% of the initial enzymic activity. In the presence of 2 mM cyanohydridoborate, a mild reducing agent which leaves intact the reacting aldehyde groups of oxidized tRNA but continuously reduces the Schiff's base in equilibrium, the activity of the enzyme can be destroyed by 100%, at a rate of 0.044 min-1, with the parallel covalent incorporation of close to one tRNA molecule per enzyme molecule. Selectivity of the labeling is also supported by the demonstration that, prior to Schiff's base formation, modified tRNA binds the transformylase with equilibrium constant and stoichiometry in good agreement with those for the active binding of unmodified tRNa. Moreover intact tRNA competes for the inactivation by the dialdehyde derivative with an affinity constant identical to that for its active binding to the enzyme