Translation termination in eukaryotes: Polypeptide release factor eRF1 is composed of functionally and structurally distinct domains

Class-1 polypeptide chain release factors (RFs) trigger hydrolysis of pepti dyl-tRNA at the ribosomal peptidyl transferase center mediated by one of th e three termination codons. In eukaryotes, apart from catalyzing the transl ation termination reaction, eRF1 binds to and activates another factor, eRF 3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidy l-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we sugges t that the two main functions of eRF1 are associated with different domains of the eRF1 protein. We show here by deletion analysis that human eRF1 is composed of two physically separated and functionally distinct domains, The "core" domain is fully competent in ribosome binding and termination-codon -dependent peptidyl-tRNA hydrolysis, and encompasses the N-terminal and mid dle parts of the polypeptide chain. The C-terminal one-third of eRF1 binds to eRF3 in vivo in the absence of the core domain, but both domains are req uired to activate eRF3 GTPase in the ribosome. The calculated isoelectric p oints of the core and C domains are 9.74 and 4.23, respectively. This highl y uneven charge distribution between the two domains implies that electrost atic interdomain interaction may affect the eRF1 binding to the ribosome an d eRF3, its activity in the termination reaction and activation of eRF3 GTP ase. The positively charged core of eRF1 may interact with negatively charg ed rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissim ilarity of the core and eRF3-binding domains implies that evolutionarily eR F1 originated as a product of gene fusion.