Three distinct peptides from the N domain of translation termination factor eRF1 surround stop codon in the ribosome

Abstract

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.

Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome

“…2). Binding of eRF1 under conditions used here is specific (i.e., occurs only if the A site is occupied with a stop codon [Bulygin et al 2002[Bulygin et al , 2010[Bulygin et al , 2011). Overall, complex 6 mimics a pretermination state, which takes place before GTP hydrolysis, whereas complex 7 corresponds to the post-termination state after GTP hydrolysis.…”

“…eRF1 is responsible for stop codon recognition and for triggering hydrolysis of the complex ester bond between the peptidyl moiety and the 3 ′ -terminal ribose of the peptidyl-tRNA located at the P site. Upon binding of eRF1 to ribosome, three highly conserved motifs of eRF1 (YxxCxxxF, TASNIKS, and GTS) located at the apex of the amino-terminal N domain mediate stop codon recognition (Bertram et al 2000;Song et al 2000;Chavatte et al 2002;Frolova et al 2002;Bulygin et al 2010Bulygin et al , 2011Conard et al 2012). Thereafter, the M domain of eRF1 binds to the peptidyl transferase center (PTC) of the ribosome inducing conformational rearrangement of the 28S rRNA and the subsequent release of the polypeptide chain (Frolova et al 1999;Song et al 2000).…”

Structure and dynamics in solution of the stop codon decoding N‐terminal domain of the human polypeptide chain release factor eRF1

“…1(C)] are all far from both Mand C-domains and therefore their large structural displacement is very likely to reflect real conformational differences between the crystal and solution states. It should be noted that loop 61-64 contains the conserved NIKS tetrapeptide sequence which is thought to be involved in the recognition of the first uridine of the stop codons 20,21 and the strictly conserved GTS loop sequence (position 31-33) which is implicated in the decoding of the stop codons. 18,20,28,29 The ability of human eRF1 to recognize each of the three stop codons UAA, UAG, and UGA and to distinguish them from the UGG tryptophan codon cannot be explained in terms of a simple static interaction.…”

“…It should be noted that loop 61-64 contains the conserved NIKS tetrapeptide sequence which is thought to be involved in the recognition of the first uridine of the stop codons 20,21 and the strictly conserved GTS loop sequence (position 31-33) which is implicated in the decoding of the stop codons. 18,20,28,29 The ability of human eRF1 to recognize each of the three stop codons UAA, UAG, and UGA and to distinguish them from the UGG tryptophan codon cannot be explained in terms of a simple static interaction. 30 It is obvious that the N-domain of human eRF1 should be able to undergo conformational rearrangements -not only in solution but also inside the ribosome.…”

“…More recently, the role of the N-domain has been confirmed unambiguously: chimeric eRF1s, in which the N-and MCdomains were derived from variant-code ciliate organisms 11,12 and human/yeast eRF1, respectively, exhibit ciliate stop codon decoding specificity in vitro. [13][14][15][16] Several hypotheses for the molecular mechanism of stop codon recognition by eRF1 have been suggested, 9,[17][18][19][20][21][22][23] but none satisfactorily explain the available experimental data which are somewhat contradictory.…”