Three distinct peptides from the N domain of translation termination factor eRF1 surround stop codon in the ribosome.
To study positioning of the polypeptide release factor eRF1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mRNA analogs, derivatives of oligoribonucleotides. The human eRF1 peptides cross-linked to these short mRNAs were identified. Cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31-33, and to lesser extent amino acids within region 121-131 (the "YxCxxxF loop") in the N domain. Hence, both regions are involved in the recognition of the purines. A cross-linker at the first uridine of the stop codon modifies Val66 near the NIKS loop (positions 61-64), and this region is important for recognition of the first uridine of stop codons. Since the N domain distinct regions of eRF1 are involved in a stop-codon decoding, the eRF1 decoding site is discontinuous and is not of "protein anticodon" type. By molecular modeling, the eRF1 molecule can be fitted to the A site proximal to the P-site-bound tRNA and to a stop codon in mRNA via a large conformational change to one of its three domains. In the simulated eRF1 conformation, the YxCxxxF motif and positions 31-33 are very close to a stop codon, which becomes also proximal to several parts of the C domain. Thus, in the A-site-bound state, the eRF1 conformation significantly differs from those in crystals and solution. The model suggested for eRF1 conformation in the ribosomal A site and cross-linking data are compatible.
Bulygin
Konstantin N
KN
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Khairulina
Yulia S
YS
Kolosov
Petr M
PM
Ven'yaminova
Aliya G
AG
Graifer
Dmitri M
DM
Vorobjev
Yuri N
YN
Frolova
Ludmila Yu
LY
Kisselev
Lev L
LL
Karpova
Galina G
GG
D001483:Base Sequence; D018388:Codon, Terminator / Q000235:genetics* / Q000378:metabolism*; D003432:Cross-Linking Reagents; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010443:Peptide Chain Termination, Translational; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism
D018388:Codon, Terminator; D003432:Cross-Linking Reagents; C489002:ETF1 protein, human; D010446:Peptide Fragments; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012343:RNA, Transfer