Ribosome-catalysed peptidyl transfer: the polyphenylalanine system

Abstract

The reaction of puromycin with polyphenylalanine-charged Escherichia coli ribosomes has been characterized. Release of polyphenylalanine from transfer RNA was estimated by a new, rapid method based on differential solubilities of substrate and product in m -cresol. Occurrence of a reaction in the absence of supernatant and GTP was demonstrated with (a) polyphenylalanine-charged 70 s ribosomes which had been washed with salt solution under conditions similar to those used for removing the supernatant factors of protein synthesis, and (b) purified, polyphenylalanine-charged 50 s subunits. Only monovalent and divalent cations and buffer are required for this “supernatant-independent” reaction, and under suitable conditions 70 to 80% of the polyphenylalanine is released from transfer RNA. Evidence from these and other results indicates that the puromycin reaction takes place by the same mechanism as peptide bond formation in protein synthesis, and that peptide bond formation is catalysed by the 50 s ribosomal subunit and does not directly involve supernatant factors or GTP. The reaction has characteristics typical of enzyme reactions, and the catalytic agent has been named “peptidyl transferase”. Kinetics of the supernatant-independent reaction are heterogeneous. A fraction of the charged ribosomes is highly reactive and may represent a preformed enzyme-substrate complex. Effects of puromycin concentration and various other factors on the kinetics are described. The reaction is inhibited by addition of 4 m -urea or 0·5% sodium dodecyl sulphate, by prior heating of charged ribosomes to 70°C, and by pre-treatment with 0·5 to 2% formaldehyde. The principle findings with 70 s ribosomes were confirmed with isolated, poly-phenylalanine-charged 50 s subunits.