[C-domain of translation termination factor eRF1 neighbors stop codon at the 80S ribosomal A site]

Positioning of stop codon and the adjacent triplet downstream of it with respect to the components of human 80S termination complex was studied with the use of mRNA analogues that bore stop signal UPuPuPu (Pu is A or G) and photoactivatable perfluoroaryl azide group. This group was attached to one of nucleotides of the stop signal or 3' of it (in positions +4 to +9 with respect to the first nucleotide of the P site codon). It was shown that upon mild UV irradiation the mRNA analogues crosslinked to components of model complexes imitating state of 80S ribosome in the course of translation termination. It was found that termination factors eRF1 and eRF3 do not affect mutual arrangement of stop signal and the 18S rRNA. Factor eRF1 was shown to cross-link to modified nucleotides in positions +5 to +9 (ability of eRF1 to cross-link to stop codon nucleotide in position +4 was shown earlier). Fragments of eRF1 containing cross-linking sites of the mRNA analogues were determined. In fragment 52-195 (containing the N-domain and a part of the M-domain) we have found cross-linking sites of the analogues that bore modifying groups on A or G in positions +5 to +9 or at the terminal phosphate of nucleotide in position +7. For mRNA analogues bearing modifying groups on G site of cross-linking from positions +5 to +7 was found in the eRF1 fragment