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Eukaryotic initiation factor 2 (eIF-2) purified from rabbit reticulocyte lysates consists of equimolar amounts of two polypeptide chains of Mr 48,000 and 38,000. Determination of the molecular weight of the native factor gave a value which is consistent with a Mr of 86,000, indicating that the factor is composed of one Mr 48,000 and one Mr 38,000 polypeptide. The purified factor exhibited all the binding activities characteristic of eIF-2. The factor formed ternary complexes with Met-tRNA? et and GTP; it bound GDP to form a binary complex; and it also possessed the property of binding a wide variety of RNA species, including reoviral mRNA, phage T3 mRNA, rRNAs, and tRNA. Furthermore, the ternary complex formed by purified eIF-2 interacted with the 40S ribosomal subunit in the presence of AUG codon to form a 40S initiation complex. These results indicate that all binding activities attributed to eIF-2 are contained in the 48,000-and 38,000-dalton polypeptides.We have shown that eukaryotic initiation factor 2 (eIF-2) purified from ribosomal salt wash proteins from calf liver is composed of one 48,000-and one 38,000-dalton polypeptide corresponding to a molecular mass of 86,000 for the native factor (1). These results were in contrast to numerous reports that eIF-2 from other eukaryotic sources, and in particular from rabbit reticulocytes, consisted of three nonidentical polypeptide chains with molecular masses in the ranges 55,000-52,000, 50,000-48,000, and 38,000-35,000 daltons (2-11). In order to resolve the anomaly in the subunit composition (and molecular mass) of eIF-2 prepared from these two mammalian sources, we purified eIF-2 from rabbit reticulocyte lysates by the procedure used for preparation of the factor from calf liver.In this paper we show that eIF-2 from rabbit reticulocytes that was purified by our rigorous purification procedure also produced only two polypeptide bands, of 48,000 and 38,000 daltons, on 15% polyacrylamide gels in the presence of sodium dodecyl sulfate. Similar to the eIF-2 from calf liver, our highly purified eIF-2 from rabbit reticulocytes exhibited all the activities known for eIF-2. MATERIALS AND METHODS Materials

Thiostrepton, a peptide antibiotic, inhibits the GTP-dependent 70S initiation complex formation (as measured by binding of fMet-tRNA to ribosomes and concomitant hydrolysis of GTP) only when initiation factor 1 is present to permit catalytic recycling of initiation factor 2 in the initiation reaction. When initiation factor 1 is absent, the binding of fMet-tRNA and GTP hydrolysis occur stoichiometrically with respect to initiation factor 2, and thiostrepton has no effect on either reaction under these conditions. Detailed analysis of this inhibition process shows that thiostrepton prevents catalytic recycling of initiation factor 2 by blocking the action of initiation factor 1, which is required for the dissociation of initiation factor 2 from the 70S initiation complex. This dissociation is necessary for the catalytic reutilization of initiation factor 2 in the initiation reaction. The antibiotic does not directly inhibit GTP hydrolysis per se in initiation.The inhibition of fMet-tRNA binding to ribosomes by thiostrepton is also dependent on the concentration of GTP; the inhibition is most pronounced at low concentrations of GTP, but at a high molar ratio of GTP to thiostrepton, the inhibition is completely abolished.Various investigators have used the peptide antibiotic, thiostrepton, in the study of ribosomal sites involved in initiation, elongation, and termination of polypeptide chains (1-12).Thiostrepton binds to the 50S ribosomal subunit and prevents ribosomal interaction with polypeptide chain elongation factors, EF-Tu and EF-G. Thiostrepton treatment of ribosomes impairs not only the binding of EF-G and the EF-G-dependent GTP hydrolysis, but also the binding of aminoacyl-tRNA (both enzymatic and nonenzymatic) and aminoacyl-tRNA EF-Tu GTP complex-dep)endent hydrolysis of GTP (5-11).These observations, together with the mutual exclusion of each factor in their binding to ribosomes (13)(14)(15)