This study is centred upon an important biological problem concerning the structural organization of mammalian ribosomes that cannot be studied by X-ray analysis because 80S ribosome crystals are still unavailable. Here, positioning of the mRNA on 80S ribosomes was studied using mRNA analogues containing the perfluorophenylazide cross-linker on either the guanosine or an uridine residue. The modified nucleotides were directed to positions from -9 to +6 with respect to the first nucleotide of the P site bound codon by a tRNA cognate to the triplet targeted to the P site. Upon mild UV-irradiation, the modified nucleotides at positions +4 to +6 cross-linked to protein S15 and 18S rRNA nucleotides A1823-A1825. In addition, modified guanosines in positions +5 and +6 also cross-linked to G626, and in position +1 to G1702. Cross-linking from the upstream positions was mainly to protein S26 that has no prokaryotic homologues. These findings indicate that the tail of mammalian S15 comes closer to the decoding site than that of its prokaryotic homologue S19, and that the environments of the upstream part of mRNA on 80S and 70S ribosomes differ. On the other hand, the results confirm the widely accepted idea regarding the conserved nature of the decoding site of the small subunit rRNA.

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.

Binding of the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA to the eIF-free 40S ribosomal subunit is the first step of initiation of translation of the viral RNA. Hairpins IIId and IIIe comprising 253–302 nt of the IRES are known to be essential for binding to the 40S subunit. Here we have examined the molecular environment of the HCV IRES in its binary complex with the human 40S ribosomal subunit. For this purpose, two RNA derivatives were used that bore a photoactivatable perfluorophenyl azide cross-linker. In one derivative the cross-linker was at the nucleotide A296 in hairpin IIIe, and in the other at G87 in domain II. Site-specific introduction of the cross-linker was performed using alkylating derivatives of oligodeoxyribonucleotides complementary to the target RNA sequences. No cross-links with the rRNA were detected with either RNA derivative. The RNA with the photoactivatable group at A296 cross-linked to proteins identified as S5 and S16 (major) and p40 and S3a (minor), while no cross-links with proteins were detected with RNA modified at G87. The results obtained indicate that hairpin IIIe is located on the solvent side of the 40S subunit head on a site opposite the beak.

The 5′-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome Entry Site) responsible for the cap-independent initiation of the viral RNA translation. At first, the IRES binds to the 40S subunit without any initiation factors so that the initiation AUG codon falls into the P site. Here using an original site-directed cross-linking strategy, we identified 40S subunit components neighboring subdomain IIId, which is critical for HCV IRES binding to the subunit, and apical loop of domain II, which was suggested to contact the 40S subunit from data on cryo-electron microscopy of ribosomal complexes containing the HCV IRES. HCV IRES derivatives that bear a photoactivatable group at nucleotide A275 or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the apex of domain II cross-link to proteins S14 and S16.

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To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3P P-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA Phe , targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3P P-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit. ß