Chavatte Laurent, Seit-Nebi Alim, Dubovaya Vera, Favre Alain
Institut Jacques Monod, UMR 7592 CNRS-Universités Paris 7-Paris 6, 2 place Jussieu, F-75251 Paris cedex 05, France.
EMBO J. 2002 Oct 1;21(19):5302-11. doi: 10.1093/emboj/cdf484.
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.
为了解析人类真核释放因子1（eRF1）在核糖体中靠近终止密码子的区域，我们使用了在终止密码子或对照有义密码子的第一位含有单个可光活化的4-硫尿苷（s(4)U）残基的mRNA。通过添加天冬氨酸tRNA（tRNA(Asp)）实现了这些mRNA在核糖体上的精确相位调整。在这些条件下，eRF1仅与含有终止密码子或s(4)UGG密码子的mRNA发生交联。我们开发了一种制备不含mRNA链的(32)P标记eRF1的方法；对野生型和突变型eRF1特异性切割后产生的标记肽段进行分析，将交联位点定位在三肽KSR（人类eRF1的63-65位），并指出位于保守NIKS环中的K63是主要交联位点。这些数据直接显示了eRF1的N端（N）结构域与40S核糖体亚基内终止密码子的相互作用，并为eRF1中间（M）结构域在60S亚基上的定位提供了有力支持。因此，N结构域和M结构域分别模拟了tRNA的反密码子臂和受体臂。
