Molecular dissection of translation termination mechanism identifies two new critical regions in eRF1.
Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomyces cerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3-a GTPase protein-probably acts as a proofreading factor, coupling stop codon recognition to polypeptide chain release. We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity. These mutations led to the identification of two new pockets in domain 1 (P1 and P2) involved in the regulation of eRF1 activity. Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1. Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein.
Hatin
Isabelle
I
Université Paris-Sud and IGM, CNRS, UMR 8621, Orsay, F 91405, France.
Fabret
Celine
C
Rousset
Jean-Pierre
JP
Namy
Olivier
O
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D017353:Gene Deletion; D005816:Genetic Complementation Test; D008958:Models, Molecular; D009154:Mutation; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D010454:Peptide Termination Factors; C080775:SUP45 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins