In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3.
Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes all three termination codons and induces hydrolysis of peptidyl tRNA, eRF3's function remains obscure. Here, we reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits; initiation, elongation, and termination factors; and aminoacyl tRNAs. This allowed us to investigate termination using pretermination complexes assembled on mRNA encoding a tetrapeptide and to propose a model for translation termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide. In this model, binding of eRF1, eRF3, and GTP to pretermination complexes first induces a structural rearrangement that is manifested as a 2 nucleotide forward shift of the toeprint attributed to pretermination complexes that leads to GTP hydrolysis followed by rapid hydrolysis of peptidyl tRNA. Cooperativity between eRF1 and eRF3 required the eRF3 binding C-terminal domain of eRF1.
Alkalaeva
Elena Z
EZ
Department of Microbiology and Immunology, SUNY Downstate Medical Center, Brooklyn, NY 11203, USA.
Pisarev
Andrey V
AV
Frolova
Lyudmila Y
LY
Kisselev
Lev L
LL
Pestova
Tatyana V
TV
D000818:Animals; D018388:Codon, Terminator; D006160:Guanosine Triphosphate / Q000502:physiology; D006868:Hydrolysis; D008954:Models, Biological; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000502:physiology; D011485:Protein Binding; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000737:chemistry; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000502:physiology; D011817:Rabbits; D012270:Ribosomes / Q000737:chemistry / Q000502:physiology
D018388:Codon, Terminator; D010454:Peptide Termination Factors; D021122:Protein Subunits; D012346:RNA, Transfer, Amino Acyl; C087971:peptide-chain-release factor 3; C019037:tRNA, peptidyl-; D006160:Guanosine Triphosphate