Recombinant subunits of mammalian elongation factor 1 expressed in Escherichia coli. Subunit interactions, elongation activity, and phosphorylation by protein kinase CKII
| Autor: | G T, Sheu, J A, Traugh |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Protein Conformation
Hydrolysis Molecular Sequence Data Protein Serine-Threonine Kinases Peptide Elongation Factors Recombinant Proteins DNA-Binding Proteins Molecular Weight Xenopus laevis Peptide Elongation Factor 1 Escherichia coli Animals Humans Amino Acid Sequence Guanosine Triphosphate Rabbits Artemia Cloning Molecular Phosphorylation Casein Kinase II |
| Zdroj: | The Journal of biological chemistry. 272(52) |
| ISSN: | 0021-9258 |
| Popis: | The first step in elongation requires two different activities; elongation factor (EF)-1alpha transfers aminoacyl-tRNA to the ribosome and is released upon hydrolysis of GTP, EF-1betagammadelta catalyzes exchange of GDP on EF-1alpha with GTP. To analyze the role of the individual subunits of EF-1 in elongation, the cDNAs for the beta, gamma, and delta subunits of EF-1 from rabbit were cloned, and proteins of 225, 437, and 280 amino acids, respectively, were expressed in Escherichia coli. The purified recombinant beta subunit migrates as a dimer and the gamma subunit as a trimer upon gel filtration, whereas the delta subunit forms a large aggregate. Complexes of betagamma, gammadelta and betagammadelta were formed by self-association and eluted with a molecular mass of approximately 160, 530, and 670 kDa, respectively; no interaction was observed between beta and delta. The activity of the recombinant subunits was determined with native EF-1alpha by measuring stimulation of the rate of elongation by poly(U)-directed polyphenylalanine synthesis. Recombinant beta and delta alone stimulated the rate of elongation by 10-fold, with a ratio of 5alpha:2beta or delta. The betagammadelta complex stimulated EF-1alpha activity up to 10-fold with a ratio of 20alpha to 1betagammadelta. Phosphorylation of the beta and delta subunits alone or in betagammadelta by protein kinase CKII had no effect on the rate of elongation. |
| Databáze: | OpenAIRE |
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