Open AccessReversal of Pactamycin Inhibition of Methionyl-Puromycin Synthesis and 80S Initiation Complex Formation by a Ribosomal Joining Factor
Author(s) -
Hideo Suzuki,
Irving H. Goldberg
Publication year - 1974
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.71.10.4259
Subject(s) - puromycin , ribosome , ribosomal rna , protein subunit , biochemistry , eukaryotic large ribosomal subunit , initiation factor , biology , protein biosynthesis , chemistry , rna , gene
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-tRNAf 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-tRNAf bound only to the small ribosomal subunit. The amount of [35 S]Met-tRNAf 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.