C-terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction

At the termination step of protein synthesis, hydrolysis of the peptidyl-tRNA is jointly catalysed at the ribosome by the termination codon and the polypeptide release factor (eRF1 in eukaryotes). eRF1 forms in vivo and in vitro a stable complex with release factor eRF3, an eRF1-dependent and riboso...

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Bibliographic Details
Published in:FEBS letters 1999-01, Vol.443 (1), p.41-47
Main Authors: Merkulova, Tatyana I., Frolova, Lyudmila Y., Lazar, Monique, Camonis, Jacques, Kisselev, Lev L.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Binding Sites
Complex formation
EF, prokaryotic elongation factor
eRF, eukaryotic polypeptide chain release factor
GAP, GTPase activating protein
GEF, guanine nucleotide exchange factor
GTP Phosphohydrolases - metabolism
Human polypeptide release factor
Humans
Interacting region
IPTG, isopropyl-1-thio-β-d-galactoside
Molecular Sequence Data
Mutation
NP40, Nonidet P40
PCR, polymerase chain reaction
PEI, polyethyleneimine
Peptide Chain Termination, Translational
Peptide Fragments - genetics
Peptide Fragments - metabolism
Peptide Termination Factors - genetics
Peptide Termination Factors - metabolism
PMSF, phenylmethylsulphonyl fluoride
PP2Ac, protein phosphatase 2A (catalytic subunit)
Protein Binding
Saccharomyces cerevisiae - genetics
Sequence Homology, Amino Acid
Species Specificity
Translation termination
X-Gal, 5-bromo-4-chloro-3-indolyl-β-d-galactoside
Yeast two-hybrid system
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Summary:At the termination step of protein synthesis, hydrolysis of the peptidyl-tRNA is jointly catalysed at the ribosome by the termination codon and the polypeptide release factor (eRF1 in eukaryotes). eRF1 forms in vivo and in vitro a stable complex with release factor eRF3, an eRF1-dependent and ribosome-dependent GTPase. The role of the eRF1⋅eRF3 complex in translation remains unclear. We have undertaken a systematic analysis of the interactions between the human eRF1 and eRF3 employing a yeast two-hybrid assay. We show that the N-terminal parts of eRF1 (positions 1–280) and of eRF3 (positions 1–477) are either not involved or non-essential for binding. Two regions in each factor are critical for mutual binding: positions 478–530 and 628–637 of eRF3 and positions 281–305 and 411–415 of eRF1. The GTP binding domain of eRF3 is not involved in complex formation with eRF1. The GILRY pentamer (positions 411–415) conserved in eukaryotes and archaebacteria is critical for eRF1's ability to stimulate eRF3 GTPase. The human eRF1 lacking 22 C-terminal amino acids remains active as a release factor and promotes an eRF3 GTPase activity whereas C-terminally truncated eRF3 is inactive as a GTPase.
ISSN:0014-5793
1873-3468
DOI:10.1016/S0014-5793(98)01669-X