The Binding of Tritiated Elongation Factors 1 and 2 to Ribosomes from Krebs II Mouse Ascites Tumor Cells

Tritiated elongation factors 1 and 2 (EF‐1 and EF‐2) were obtained from Krebs II ascites cells which had been grown in mice injected with radioactive amino acids. The highly purified factors were sufficiently radioactive to be used in a study of the interactions between ribosomes and elongation factors.The following results were obtained. EF‐1 binding to ribosomes requires the presence of a polynucleotide, an aminoacyl‐tRNA specified by the latter and a guanosine nucleotide carrying three phosphate groups. The hydrolysis of the GTP molecule involved in the binding reaction leads to the immediate release of EF‐1. If GTP is replaced by Guo‐5′‐P2‐CH2‐P the factor remains bound to the ribosome and can be detected by sucrose gradient centrifugation techniques. Likewise EF‐2 binding to ribosomes can only be detected in the presence of Guo‐5′‐P2‐CH2‐P. The affinity of ribosomes for EF‐2 appears to be higher than for EF‐1: preincubation of ribosomes with EF‐2 inhibits the subsequent attachment of EF‐1 almost completely. EF‐1 prebound to ribosomes in the presence of Guo‐5′‐P2‐CH2‐P, poly(uridy1ic acid) and Phe‐tRNAPhe is partially removed from the ribosomes together with Phe‐tRNA during a second incubation with EF‐2. Although EF‐2 binding to ribosomes precludes any stable association between ribosomes and EF‐1 it does not prevent the insertion of aminoacyl‐tRNA into the ribosomal A‐site. The attachment of aminoacyl‐tRNA under these conditions enhances the binding of EF‐2 to the ribosome. The antibiotic showdomycin strongly inhibits the attachment of EF‐1 to ribosomes and to a lesser degree impairs the binding of EF‐2. A‐site ribosomes display a strong preference for the attachment of EF‐2 and bind EF‐1 only very poorly. The reverse is true for P‐site ribosomes which are good substrates for the binding of EF‐1 and bind EF‐2 less efficiently than A‐site ribosomes.These results and a number of additional findings made in this and in previous studies are discussed in the general context of the structure and function of mammalian elongation factors 1 and 2.