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The invariant uridine of stop codons contacts the conserved NIKSR loop of human eRF1 in the ribosome
Author(s) -
Chavatte Laurent,
SeitNebi Alim,
Dubovaya Vera,
Favre Alain
Publication year - 2002
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/cdf484
Subject(s) - biology , stop codon , release factor , ribosome , transfer rna , eukaryotic ribosome , ribosomal rna , genetics , rna , biochemistry , gene
To unravel the region of human eukaryotic release factor 1 (eRF1) that is close to stop codons within the ribosome, we used mRNAs containing a single photoactivatable 4‐thiouridine (s 4 U) residue in the first position of stop or control sense codons. Accurate phasing of these mRNAs onto the ribosome was achieved by the addition of tRNA Asp . Under these conditions, eRF1 was shown to crosslink exclusively to mRNAs containing a stop or s 4 UGG codon. A procedure that yielded 32 P‐labeled eRF1 deprived of the mRNA chain was developed; analysis of the labeled peptides generated after specific cleavage of both wild‐type and mutant eRF1s maps the crosslink in the tripeptide KSR (positions 63–65 of human eRF1) and points to K63 located in the conserved NIKS loop as the main crosslinking site. These data directly show the interaction of the N‐terminal (N) domain of eRF1 with stop codons within the 40S ribosomal subunit and provide strong support for the positioning of the eRF1 middle (M) domain on the 60S subunit. Thus, the N and M domains mimic the tRNA anticodon and acceptor arms, respectively.