Arrangement of the Sense and Stop Codons of the Template in the A Site of the Human Ribosome as Inferred from Crosslinking with Oligonucleotide Derivatives

Oligoribonucleotide derivatives containing Phe codon UUC along with a 3-flanking sense or stop codon with a perfluoroarylazido group at G or U were used to study the positioning of each nucleotide of the latter codon relative to the 18S rRNA in the A site of the 80S ribosome. To place the modified sense or stop codon in the A site, tRNA^Phe cognate to UCC was bound in the P site. Regardless of the position in the sense or stop codon, the modified nucleotide crosslinked with invariant dinucleotide A1823/A1824 and nucleotide A1825 in helix 44 close to the 3 end of the 18S rRNA. Located in the second or third position of either codon, the modified G bound with invariant nucleotide G626, which is in the evolutionarily conserved 530 stem–loop fragment. The results were collated with the X-ray structure of the bacterial ribosome, and the template codon was assumed to be similarly arranged relative to the small-subunit rRNA in the ribosomal A site of various organisms.     

Variable and conserved elements of human ribosomes surrounding the mRNA at the decoding and upstream sites

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 modi-fied 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.     

Template Location on the Human Ribosome: Environment of the mRNA Nucleotide Adjacent to the A-Site Codon on the 3′-Side

The 18S rRNA nucleotides close to the template nucleotide adjacent to the 80S ribosomal A-site codon on the 3′-end (i.e., the nucleotide in position +7 relative to the first nucleotide of the P-site codon) were identified using the affinity crosslinking approach. For this purpose, the photoreactive mRNA analogues with a perfluorophenylazide group attached through various linkers to the uridine C5, 3′-terminal phosphate or guanosine N7 were used. The position of the mRNA analogues on the ribosome was preset using tRNA^Phe, which recognized the phenylalanine codon directed to the P-site. An analysis of the rRNAs isolated from the irradiated complexes of 80S ribosomes showed that all the analogues are almost equally crosslinked to the 18S rRNA nucleotides we attributed to the A-site codon environment: namely, to nucleotides A1823, A1824, and A1825 of the 3′-minidomain and to the 620–630 fragment of the 18S rRNA 5′-domain. In addition, we identified a new component of the mRNA binding site of human ribosomes, nucleotide C1698, belonging to the 18S rRNA 3′-minidomain, using analogues bearing a perfluorophenylazide group on uridine and guanine residues.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 295–302.Original Russian Text Copyright © 2005 by Demeshkina, Styazhkina, Bulygin, Repkova, Ven’yaminova, Karpova.