Reaction of puromycin with chemically prepared peptidyl transfer RNA.

Abstract

Chemically synthesized peptidyl‐tRNAphe reacts with puromycin when bound non‐enzymatically to Escherichia coli ribosomes in the presence of poly U. Guanosine triphosphate and G factor have no influence upon the puromycin reaction, when the magnesium ion concentration is 0.01 M or lower, but stimulate the puromycin reaction when the binding of peptidyl‐tRNA is carried out at higher magnesium ion concentration. In this stimulation β,γ‐methylene‐guanosine triphosphate cannot replace GTP, and fusidic acid inhibits the stimulation. The percentage of bound peptidyl‐tRNA which reacts with puromycin in the absence of G factor and GTP decreases upon increasing the magnesium ion concentration. From all these observations one can conclude that at magnesium ion concentrations below 0.01 M, peptidyl‐tRNA (such as Gly2‐Phe‐tRNA) binds preferentially to the donor site, but at higher magnesium ion concentrations (such as 0.025 M) the ribosomal aceptor site is preferred.Peptidyl‐tRNAPhe can react with puromycin in the absence of the proper mRNA at rather highmagnesium ion concentration (0.02 M and higher) but this interaction in the absence of mRNA may be not a common feature of other peptidyl‐tRNAs, except tRNAMetf.Phe‐tRNA interacts with puromycin but the rate of this interaction is much slower than that of peptidyl‐tRNAPhe such as Gly2‐Phe‐tRNA.