Abstract
During termination of translation in eukaryotes, a GTP-binding protein, eRF3, functions within a complex with the tRNA-mimicking protein, eRF1, to decode stop codons. It remains unclear how the tRNA-mimicking protein co-operates with the GTPase and with the functional sites on the ribosome. In order to elucidate the molecular characteristics of tRNA-mimicking proteins involved in stop codon decoding, we have devised a heterologous genetic system in Saccharomyces cerevisiae. We found that eRF3 from Pneumocystis carinii (Pc-eRF3) did not complement depletion of S. cerevisiae eRF3. The strength of Pc-eRF3 binding to Sc-eRF1 depends on the GTP-binding domain, suggesting that defects of the GTPase switch in the heterologous complex causes the observed lethality. We isolated mutants of Pc-eRF3 and Sc-eRF1 that restore cell growth in the presence of Pc-eRF3 as the sole source of eRF3. Mapping of these mutations onto the latest 3D-complex structure revealed that they were located in the binding-interface region between eRF1 and eRF3, as well as in the ribosomal functional sites. Intriguingly, a novel functional site was revealed adjacent to the decoding site of eRF1, on the tip domain that mimics the tRNA anticodon loop. This novel domain likely participates in codon recognition, coupled with the GTPase function.
Highlights
Termination of translation in eukaryotes is catalyzed by two classes of polypeptide release factors, eRF1 and eRF3 [1,2,3]. eRF1 recognizes stop codons directly and activates mature polypeptide release by peptidyl-tRNA hydrolysis [4,5,6]. eRF3 is a G-protein that is related to translation elongation factors (EFs), and stimulates polypeptide release by eRF1 [7,8]. eRF1 and eRF3 heterodimerize
Similar to other eRF3 orthologs, the eRF3 derived from P. carinii (Pc-eRF3) contains a less-conserved N-terminal region [1,9,18], which is thought to be dispensable for the eRF3 (SUP35) defective yeast cell growth complementation [1,43]
Expression plasmids were introduced into the conditional-lethal eRF3 S. cerevisiae strain, Y40, in which the endogenous eRF3 promoter is replaced with the tet-OFF promoter, which can be deactivated in the presence of doxycycline
Summary
Termination of translation in eukaryotes is catalyzed by two classes of polypeptide release factors, eRF1 (class I) and eRF3 (class II) [1,2,3]. eRF1 recognizes stop codons directly and activates mature polypeptide release by peptidyl-tRNA hydrolysis [4,5,6]. eRF3 is a G-protein that is related to translation elongation factors (EFs), and stimulates polypeptide release by eRF1 [7,8]. eRF1 and eRF3 heterodimerize. ERF1 recognizes stop codons directly and activates mature polypeptide release by peptidyl-tRNA hydrolysis [4,5,6]. This binding between eRF1 and eRF3 has been studied extensively [1,2,9,10], and the C-terminal domains of both eRF1 and eRF3 were shown to be sufficient for this interaction. Domain M contains the universally conserved GGQ motif that corresponds to the CCA-end of tRNA [4,5,16] and plays a role in peptide release from the peptidyl-tRNA by activating hydrolysis at the peptidyl transferase center of the ribosome [14]
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