Exploring accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel at various steps of translation initiation.

In this work, we studied how the accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel, ribosomal protein (rp) uS3 and helix (h) 16 of the 18S rRNA, changes upon the translation initiation. In particular, we examined the accessibility of rp uS3 for binding of unstructured RNAs and of riboses in h16 towards attack with benzoyl cyanide (BzCN) in complexes assembled in rabbit reticulocyte lysate utilizing synthetic oligoribonucleotides as well as full-length and truncated up to the initiation AUG codon hepatitis C virus IRES as model mRNAs. With both mRNA types, the rp uS3 peptide recognizing single-stranded RNAs was shown to become shielded only in those 48S preinitiation complexes (PICs) that contained eIF3j bound to 40S subunit in the area between the decoding site and the mRNA entry channel.

Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome.

Translation termination in eukaryotes is mediated by release factors: eRF1, which is responsible for stop codon recognition and peptidyl-tRNA hydrolysis, and GTPase eRF3, which stimulates peptide release. Here, we have utilized ribose-specific probes to investigate accessibility of rRNA backbone in complexes formed by association of mRNA- and tRNA-bound human ribosomes with eRF1•eRF3•GMPPNP, eRF1•eRF3•GTP, or eRF1 alone as compared with complexes where the A site is vacant or occupied by tRNA. Our data show which rRNA ribose moieties are protected from attack by the probes in the complexes with release factors and reveal the rRNA regions increasing their accessibility to the probes after the factors bind.

Molecular contacts of ribose-phosphate backbone of mRNA with human ribosome.

In this work, intimate contacts of riboses of mRNA stretch from nucleotides in positions +3 to +12 with respect to the first nucleotide of the P site codon were studied using cross-linking of short mRNA analogs with oxidized 3'-terminal riboses bound to human ribosomes in the complexes stabilized by codon-anticodon interactions and in the binary complexes. It was shown that in all types of complexes cross-links of the mRNA analogs to ribosomal protein (rp) uS3 occur and the yield of these cross-links does not depend on the presence of tRNA and on sequences of the mRNA analogs. Site of the mRNA analogs cross-linking in rp uS3 was mapped to the peptide in positions 55-64 that is located away from the mRNA binding site.

Ribosomal protein S5e is implicated in translation initiation through its interaction with the N-terminal domain of initiation factor eIF2α.

A key step of translation initiation in eukaryotes is formation of the 48S preinitiation complex (PIC) containing the 40S ribosome, a set of eukaryotic initiation factors (eIFs), mRNA, and initiator Met-tRNA interacting with mRNA start codon; however, the PIC structure remains substantially unknown. Here, we apply formaldehyde-induced protein-protein crosslinks to identify contacts between ribosomal protein S5e (rpS5e, "e" stands for "eukaryotic") and eIFs within the mammalian PIC, assembled on either model canonical or IRES-containing mRNA. Using immunoblotting and mass spectrometry, we show that with both types of mRNA, rpS5e crosslinks to eIF2α.