Termination of translation in eukaryotes is mediated by the quaternary eRF1*eRF3*GTP*Mg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct.
GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg2+. We show that (i) eRF3 binds GDP (K(d) = 1.9 microM) and this interaction depends only minimally on the Mg(2+) concentration; (ii) GTP binds to eRF3 (K(d) = 0.5 microM) only in the presence of eRF1 and this interaction depends on the Mg2+ concentration; (iii) GTP displaces GDP from the eRF1*eRF3*GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1*eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1*eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1*eRF3*GTP*Mg2+, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3*eRF1 complex profoundly differ from those of prokaryotic RF3.
Mitkevich
Vladimir A
VA
Engelhardt Institute of Molecular Biology, the Russian Academy of Sciences, Moscow 119991, Russia.
Kononenko
Artem V
AV
Petrushanko
Irina Yu
IY
Yanvarev
Dmitry V
DV
Makarov
Alexander A
AA
Kisselev
Lev L
LL
D001426:Bacterial Proteins / Q000502:physiology; D002151:Calorimetry; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000378:metabolism*; D008954:Models, Biological; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000378:metabolism* / Q000502:physiology*
D001426:Bacterial Proteins; C489002:ETF1 protein, human; D010454:Peptide Termination Factors; C087971:peptide-chain-release factor 3; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D008274:Magnesium