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. 1. 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. 2. Likewise EF-2 binding to ribosomes can only be detected in the presence of GUO-5'-P2-CH2-P. 3. 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(URIDYLIC ACID) AND Phe-tRNA-Phe is partially removed from the ribosomes together with Phe-tRNA during a second incubation with EF-2. 4. 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. 5. The antibiotic showdomycin strongly inhibits the attachment of EF-1 to ribosomes and to a lesser degree impairs the binding of EF-2. 6. 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.
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D002287:Carcinoma, Krebs 2 / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006150:Guanine Nucleotides / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010445:Peptide Elongation Factors; D010649:Phenylalanine / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D011485:Protein Binding; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium
D000900:Anti-Bacterial Agents; D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; D014316:Tritium; D010649:Phenylalanine