Recombinant subunits of mammalian elongation factor 1 expressed in Escherichia coli. Subunit interactions, elongation activity, and phosphorylation by protein kinase CKII.
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.
Sheu
G T
GT
Department of Biochemistry and the Genetics Graduate Group, University of California, Riverside, California 92521-0129, USA.
Traugh
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001156:Artemia; D047390:Casein Kinase II; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D014982:Xenopus laevis
D004268:DNA-Binding Proteins; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases