GTP-dependent structural rearrangement of the eRF1:eRF3 complex and eRF3 sequence motifs essential for PABP binding.
Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation.
Kononenko
Artem V
AV
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow 119991, Russia.
Mitkevich
Vladimir A
VA
Atkinson
Gemma C
GC
Tenson
Tanel
T
Dubovaya
Vera I
VI
Frolova
Ludmila Yu
LY
Makarov
Alexander A
AA
Hauryliuk
Vasili
V
D020816:Amino Acid Motifs; D001665:Binding Sites; D019295:Computational Biology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D016296:Mutagenesis; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013696:Temperature
C489002:ETF1 protein, human; D010454:Peptide Termination Factors; D039102:Poly(A)-Binding Proteins; C087971:peptide-chain-release factor 3; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate