Structural and Functional Topography of Human Ribosomes Deduced from Crosslinking with mRNA Analogs,Oligoribonucleotide Derivatives

Reviewed are data on the position of template codons with respect to 18S rRNA and certain proteins on human ribosome obtained using a set of mRNA analogs, oligoribonucleotide derivatives carrying alkylating or photoactivatable groups at different positions. A comparison of data on the template position on the human and Escherichia coliribosomes has revealed both the similarity in the structure of the mRNA-binding site of bacterial and mammalian ribosomes and the peculiarities of the functioning of mammalian (in particular, human) ribosomes. The similarity manifests itself in that the template codons at the A-, P-, and E-sites of bacterial and human ribosomes are surrounded by similar nucleotides (occupying similar positions in the conserved regions of secondary structure) of small subunit rRNA. The template forms a loop whose foot is in proximity to the 530 stem–loop conserved region of rRNA. The specific features of mammalian ribosomes appear to be associated with their lower conformational mobility as compared with bacterial ribosomes, owing to which their interaction with the template involves a lesser number of molecular contacts.     

Region 1112–1123 in the central domain of 18S rRNA in 40S subunits of plant ribosomes: Accessibility for complementary interactions and the functional role

The binding of the 18S rRNA of the 40S subunits of wheat germ ribosomes to an oligodeoxyribonucleotide complementary to the 1112–1123 region of the central domain of this RNA molecule has been studied. The selective binding of this oligomer to the complementary RNA fragment and the inhibition of the translation of uncapped chimeric RNA containing enhancer sequences in the 5′-untranslated region upstream of the reporter sequence coding for β-glucuronidase has been shown in a cell-free protein-synthesizing system. The use of a derivative of the aforementioned oligomer containing an alkylating group at the 5′ end allowed for the demonstration that the 1112–1123 region of 18S RNA can form a heteroduplex with the complementary sequence of the oligomer. The data obtained show that the 1112–1123 region in loop 27 of the central domain of 18S RNA of 40S ribosomal subunits is exposed on the subunit surface and probably participates in the cap-independent binding of the subunits to mRNA due to the complementary interaction with the enhancer sequences.     

Ribosomal protein uS3 in cell biology and human disease: Latest insights and prospects

The conserved ribosomal protein uS3 in eukaryotes has long been known as one of the essential components of the small (40S) ribosomal subunit, which is involved in the structure of the 40S mRNA entry pore, ensuring the functioning of the 40S subunit during translation initiation. Besides, uS3, being outside the ribosome, is engaged in various cellular processes related to DNA repair, NF-kB signaling pathway and regulation of apoptosis. This review is devoted to recent data opening new horizons in understanding the roles of uS3 in such processes as the assembly and maturation of 40S subunits, ensuring proper structure of 48S pre-initiation complexes, regulation of initiation and ribosome-based RNA quality control pathways. Besides, we summarize novel results on the participation of the protein in processes beyond translation and consider biomedical implications of previously known and recently found extra-ribosomal functions of uS3, primarily, in oncogenesis.     

The C-terminal fragment of ribosomal protein S15 is located in the decoding site of the human ribosome

Protein S15 is a characteristic component of the mammalian 80S ribosome that neighbors the mRNA codon at the decoding site and the downstream triplets. The S15 fragment juxtaposed in the human ribosome to mRNA nucleotides +4 to +12 relative to the first nucleotide of the P-site codon was determined. S15 was modified using a set of mRNA analogs containing the triplet UUU/UUC at the 5′ end and a perfluorophenyl azide-carrying uridine at various positions downstream of this triplet. The mRNA analogs were positioned on the ribosome with the use of tRNA^Phe, cognate to the UUU/UUC triplet, targeted to the P site. Modified S15 was isolated from complexes of 80S ribosomes with tRNA^Phe and the mRNA analogs after irradiation with mild UV light and hydrolyzed with cyanogen bromide, cleaving the polypeptide chain after Met residues. Analysis of the modified oligopeptides resulting from hydrolysis demonstrated that the crosslinking site was in C-terminal fragment 111–145 of S15 in all cases, suggesting the involvement of this fragment in the decoding site of the eukaryotic ribosome.     

Highly Conserved Region 1816–1831 of the 18S Ribosomal RNA Is Close to the First Nucleotide of the A-Site Codon in Elongation and Termination of Translation

Two mRNA analogs, pUUCUAAA (with stop codon UAA) and pUUCUCAA (with Ser codon UCA) containing a perfluoroarylazido group at U4, were used to study the position relative to the 18S rRNA for the first nucleotide of the codon located in the A site of the human 80S ribosome. To place UAA or UCA in the A site, UCC-recognizing tRNA^Phe was bound in the P site. With each analog, crosslinking was detected for highly conserved fragment 1816–1831, which contains invariant dinucleotide A1823/A1824 and is in helix 44 at the 3" end of the 18S rRNA. Since 18S rRNA modification did not depend on whether the U4 photoreactive group was in the sense or stop codon, it was assumed that polypeptide chain release factor 1 directly recognizes the trinucleotide of a stop codon located in the A site.     

The C domain of translation termination factor eRF1 is close to the stop codon in the A site of the 80S ribosome

The arrangement of the stop codon and its 3′-flanking codon relative to the components of translation termination complexes of human 80S ribosomes was studied using mRNA analogs containing the stop signal UPuPuPu (Pu is A or G) and the photoreactive perfluoroarylazido group, which was linked to a stop-signal or 3′-flanking nucleotide (positions from +4 to +9 relative to the first nucleotide of the P-site codon). Upon mild UV irradiation, the analogs crosslinked to components of the model complexes, mimicking the state of the 80S ribosome at translation termination. Termination factors eRF1 and eRF3 did not change the relative arrangement of the stop signal and 18S rRNA. Crosslinking to eRF1 was observed for modified nucleotides in positions +5 to +9 (that for stop-codon nucleotide +4 was detected earlier). The eRF1 fragments crosslinked to the mRNA analogs were identified. Fragment 52–195, including the N domain and part of the M domain, crosslinked to the analogs carrying the reactive group at A or G in positions +5 to +9 or at the terminal phosphate of nucleotide +7. The site crosslinking to mRNA analogs containing modified G in positions +5 to +7 was assigned to eRF1 fragment 82–166 (beyond the NIKS motif). All but one analog (that with modified G in position +4) crosslinked to the C domain of eRF1 (fragment 330–422). The efficiency of crosslinking to the C domain was higher than to the N domain in most cases. It was assumed that the C domain of eRF1 bound in the A site is close to nucleotides +5 to +9, especially +7 and +8, and that eRF1 undergoes substantial conformational changes when binding to the ribosome.     

Adenine and guanine recognition of stop codon is mediated by different N domain conformations of translation termination factor eRF1

Positioning of release factor eRF1 toward adenines and the ribose-phosphate backbone of the UAAA stop signal in the ribosomal decoding site was studied using messenger RNA (mRNA) analogs containing stop signal UAA/UAAA and a photoactivatable cross-linker at definite locations. The human eRF1 peptides cross-linked to these analogs were identified. Cross-linkers on the adenines at the 2nd, 3rd or 4th position modified eRF1 near the conserved YxCxxxF loop (positions 125-131 in the N domain), but cross-linker at the 4th position mainly modified the tripeptide 26-AAR-28. This tripeptide cross-linked also with derivatized 3'-phosphate of UAA, while the same cross-linker at the 3'-phosphate of UAAA modified both the 26-28 and 67-73 fragments. A comparison of the results with those obtained earlier with mRNA analogs bearing a similar cross-linker at the guanines indicates that positioning of eRF1 toward adenines and guanines of stop signals in the 80S termination complex is different. Molecular modeling of eRF1 in the 80S termination complex showed that eRF1 fragments neighboring guanines and adenines of stop signals are compatible with different N domain conformations of eRF1. These conformations vary by positioning of stop signal purines toward the universally conserved dipeptide 31-GT-32, which neighbors guanines but is oriented more distantly from adenines.     

Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit

The molecular environment of the internal ribosome entry site (IRES element) of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group at nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the subunit between the head and the body aside the “beak” near the exit from the mRNA-binding channel.