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

Published online by Cambridge University Press:  01 March 2000

LUDMILA YU. FROLOVA
Affiliation:
Engelhardt Institute of Molecular Biology, 117984 Moscow, Russia U248 Institut Nationale de la Santé et de la Recherche Médicale, Institut Curie, Paris 70005, France UPR41 Centre National de la Recherche Scientifique, Universite de Rennes 1, 350042 Rennes Cedex, France
TATYANA I. MERKULOVA
Affiliation:
U248 Institut Nationale de la Santé et de la Recherche Médicale, Institut Curie, Paris 70005, France
LEV L. KISSELEV
Affiliation:
Engelhardt Institute of Molecular Biology, 117984 Moscow, Russia U248 Institut Nationale de la Santé et de la Recherche Médicale, Institut Curie, Paris 70005, France
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Abstract

Class-1 polypeptide chain release factors (RFs) trigger hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center mediated by one of the three termination codons. In eukaryotes, apart from catalyzing the translation termination reaction, eRF1 binds to and activates another factor, eRF3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidyl-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we suggest 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 middle 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 required to activate eRF3 GTPase in the ribosome. The calculated isoelectric points of the core and C domains are 9.74 and 4.23, respectively. This highly uneven charge distribution between the two domains implies that electrostatic interdomain interaction may affect the eRF1 binding to the ribosome and eRF3, its activity in the termination reaction and activation of eRF3 GTPase. The positively charged core of eRF1 may interact with negatively charged rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissimilarity of the core and eRF3-binding domains implies that evolutionarily eRF1 originated as a product of gene fusion.

Type
Research Article
Copyright
2000 RNA Society

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