Reversal of pactamycin inhibition of methionyl-puromycin synthesis and 80S initiation complex formation by a ribosomal joining factor.

Abstract

A crude mixture of polypeptide chain initiation factors (0.5 M KCl ribosomal wash) from reticulocyte ribosomes was fractionated by DEAE-cellulose column chromatography. Among several initiation factors obtained from the column, one factor eluting at 0.22-0.25 M KCl showed a remarkable ability to overcome the inhibition of Met-puromycin and 80S initiation complex formation caused by the antibiotic, pactamycin. Earlier experiments had shown that pactamycin does not prevent the binding of Met-tRNA(f) to the small ribosomal subunit but does interfere with the joining of the 60S ribosomal subunit to form the 80S initiation complex. A Lineweaver-Burk plot of initial rates of Met-puromycin formation showed that the interaction of the factor and pactamycin was of a competitive type. In the absence of the factor, [(35)S]Met-puromycin was not synthesized and [(35)S]Met-tRNA(f) bound only to the small ribosomal subunit. The amount of [(35)S]Met-tRNA(f) bound to 80S ribosomes bearing endogenous mRNA and the amount of [(35)S]Met-puromycin formed were directly related to the amount of factor added. Thus, this factor can be termed a "joining factor," and a simple assay of its activity can be devised based on its ability to overcome the pactamycin inhibition of the puromycin reaction.