New insights into stop codon recognition by eRF1.
In eukaryotes, translation termination is performed by eRF1, which recognizes stop codons via its N-terminal domain. Many previous studies based on point mutagenesis, cross-linking experiments or eRF1 chimeras have investigated the mechanism by which the stop signal is decoded by eRF1. Conserved motifs, such as GTS and YxCxxxF, were found to be important for termination efficiency, but the recognition mechanism remains unclear. We characterized a region of the eRF1 N-terminal domain, the P1 pocket, that we had previously shown to be involved in termination efficiency. We performed alanine scanning mutagenesis of this region, and we quantified in vivo readthrough efficiency for each alanine mutant. We identified two residues, arginine 65 and lysine 109, as critical for recognition of the three stop codons. We also demonstrated a role for the serine 33 and serine 70 residues in UGA decoding in vivo. NMR analysis of the alanine mutants revealed that the correct conformation of this region was controlled by the YxCxxxF motif. By combining our genetic data with a structural analysis of eRF1 mutants, we were able to formulate a new model in which the stop codon interacts with eRF1 through the P1 pocket.
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 400, 91400 Orsay, France.
Sandra
S
Blanchet
School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK.
Michelle
M
Rowe
School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK.
Tobias
T
Von der Haar
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 400, 91400 Orsay, France.
Céline
C
Fabret
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 400, 91400 Orsay, France.
Stéphane
S
Demais
School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK.
Mark J
MJ
Howard
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 400, 91400 Orsay, France olivier.namy@igmors.u-psud.fr.
Olivier
O
Namy
D018388:Codon, Terminator; D008958:Models, Molecular; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018388:Codon, Terminator; C489002:ETF1 protein, human; D010454:Peptide Termination Factors; D011994:Recombinant Proteins; C080775:SUP45 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins