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.

Previously we have shown that the CCA end of a P-tRNA can be crosslinked with the RPL36AL protein of the large subunit of mammalian ribosomes; it belongs to the L44e protein family present in all eukaryotic and archaeal ribosomes. Here we confirm and extend this finding and demonstrate that: 1) this crosslink is specific for a tRNA at the P/E hybrid site, as a tRNA in all other tRNA positions of pre-translocational ribosomes could not be crosslinked with a ribosomal protein, 2) the crosslink was formed most efficiently with C74 and C75 of P/E-tRNA, but could also connect the ultimate A of this tRNA with Lys53 of protein RPL36AL, 3) this protein contains seven monomethylated residues (three lysyl and three arginyl residues, as well as glutaminyl residue 51), 4) Q51 is part of a conserved GGQ motif in the L44e proteins in eukaryotic 80S ribosomes that is identical to the universally conserved motif of release factors implicated in promoting peptidyl-tRNA hydrolysis, and 5) the large number of modifications, in which some of the residues were methylated to about 50 %, might indicate that protein RPL36AL is a preferential target for regulation.