Nucleotide-mediated interactions of eukaryotic elongation factor EF-2 with ribosomes.

Eukaryotic elongation factor EF-2 isolated from rat liver microsomal salt-wash showed two types of nucleotide-dependent interactions with reconstituted empty 80S ribosomes when analyzed by gradient centrifugation. Stable EF-2 X ribosome complexes were only formed with GTP analogues with reduced or no ability to serve as substrates for the EF-2 and ribosome-dependent GTPase. GTP-stimulated complex formation was only demonstrable after glutaraldehyde fixation, unless a GTP-regenerating system was included throughout the gradient centrifugation. GDP and to a lesser extent GMP and guanosine also stimulated the less stable type of complex formation as demonstrable after fixation. Under the same conditions some complex formation was also observed with ATP and its analogue adenosine 5'-[beta,gamma-methylene]triphosphate, although with less efficiency than with the corresponding guanosine nucleotides. The results in combination with available data indicate that EF-2 has two binding states with different affinities on the 80S ribosome: a high-affinity pre-translocation state specific for EF-2 X GTP and a low-affinity post-translocation state, in which EF-2 X GDP is bound to the ribosome in a less stable and specific complex.