Comparative Analysis of Ribosome-Associated Adenosinetriphosphatase (ATPase) from Pig Liver and the ATPase of Elongation Factor 3 from Saccharomyces cerevisiae

Abstract

Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational machinery. Non-fungal organisms do not have and do not require a soluble form of the third elongation factor for translation. To test whether non-fungal organisms have a direct analog of EF-3 incorporated in the structure of the ribosomes, a comparison of EF-3 adenosinetriphosphatase (ATPase) with ATPases associated with pig liver ribosomes was carried out. EF-3 function depends on ATP (GTP) hydrolysis. The hydrolytic activity of EF-3 is enhanced by two orders of magnitude by yeast ribosomes due to an increase in the turnover rate of EF-3. The nucleotide hydrolytic activity of EF-3 is significantly constrained by the binding of aminoacylated tRNA(Phe) to poly(U)-programmed ribosomes. The translational inhibitors--neomycin and alpha-sarcin suppress the ATPase activity of EF-3. These results reflect a direct correlation between EF-3 ATPase and the functional state of the ribosome. Four lines of evidence indicate that yeast EF-3 ATPase is functionally distinct from pig liver ribosome associated ATPases. The kinetic parameters of ATPases from these two sources are different. Poly(U) and tRNA have no effect on the ATPase activity associated with the pig liver ribosomes. The latter activity is unaffected by the translational inhibitors neomycin and alpha-sarcin. The translational activity of pig liver ribosomes is not influenced by ATP, ADP or adenosine 5'-[beta, gamma-imido]triphosphate. In an in vitro system, one can demonstrate a small but consistent stimulatory effect of yeast EF-3 on polyphenylalanine synthesis by pig liver ribosomes only when EF-1 alpha is present at a limited concentration. The EF-3 effect disappears when EF-1 alpha is added in a stoichiometric amount to the pig liver ribosomes. This result is in contrast to the yeast system where the ribosomes are completely dependent on EF-3 at all concentrations of EF-1 alpha.