Invariant amino acids essential for decoding function of polypeptide release factor eRF1.
In eukaryotic ribosome, the N domain of polypeptide release factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125-131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains.
Kolosov
Petr
P
Engelhardt Institute of Molecular Biology, the Russian Academy of Sciences, 119991 Moscow, Russia.
Frolova
Ludmila
L
Seit-Nebi
Alim
A
Dubovaya
Vera
V
Kononenko
Artem
A
Oparina
Nina
N
Justesen
Just
J
Efimov
Alexandr
A
Kisselev
Lev
L
D000595:Amino Acid Sequence; D018388:Codon, Terminator; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
D018388:Codon, Terminator; C489002:ETF1 protein, human; D010454:Peptide Termination Factors; D018698:Glutamic Acid; D014443:Tyrosine