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Previous analyses of complexes of 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by the IRES with ribosomal proteins. Here, using chemical probing, we show that the HCV IRES also contacts the backbone and bases of the CCC triplet in the 18S ribosomal RNA (rRNA) expansion segment 7. These contacts presumably provide interplay between IRES domain II and the AUG codon close to ribosomal protein S5, which causes a rearrangement of 18S rRNA structure in the vicinity of the universally conserved nucleotide G1639. As a result, G1639 becomes exposed and the corresponding site of the 40S subunit implicated in transfer RNA discrimination can select . These data are the first demonstration at nucleotide resolution of direct IRES–rRNA interactions and how they induce conformational transition in the 40S subunit allowing the HCV IRES to function without AUG recognition initiation factors.

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SBP2 is a pivotal protein component in selenoprotein synthesis. It binds the SECIS stem-loop in the 3 ′ UTR of selenoprotein mRNA and interacts with both the specialized translation elongation factor and the ribosome at the 60S subunit. In this work, our goal was to identify the binding partners of SBP2 on the ribosome. Cross-linking experiments with bifunctional reagents demonstrated that the SBP2-binding site on the human ribosome is mainly formed by the 28S rRNA. Direct hydroxyl radical probing of the entire 28S rRNA revealed that SBP2 bound to 80S ribosomes or 60S subunits protects helix ES7L-E in expansion segment 7 of the 28S rRNA. Diepoxybutane cross-linking confirmed the interaction of SBP2 with helix ES7L-E. Additionally, binding of SBP2 to the ribosome led to increased reactivity toward chemical probes of a few bases in ES7L-E and in the universally conserved helix H89, indicative of conformational changes in the 28S rRNA in response to SBP2 binding. This study revealed for the first time that SBP2 makes direct contacts with a discrete region of the human 28S rRNA.

The amino acid selenocysteine is encoded by UGA, usually a stop codon, thus requiring a specialized machinery to enable its incorporation into selenoproteins. The machinery comprises the tRNA Sec , a 3 ′ -UTR mRNA stem-loop termed SElenoCysteine Insertion Sequence (SECIS), which is mandatory for recoding UGA as a Sec codon, the SECIS Binding Protein 2 (SBP2), and other proteins. Little is known about the molecular mechanism and, in particular, when, where, and how the SECIS and SBP2 contact the ribosome. Previous work by others used the isolated SECIS RNA to address this question. Here, we developed a novel approach using instead engineered minimal selenoprotein mRNAs containing SECIS elements derivatized with photoreactive groups. By cross-linking experiments in rabbit reticulocyte lysate, new information could be gained about the SBP2 and SECIS contacts with components of the translation machinery at various translation steps. In particular, we found that SBP2 was bound only to the SECIS in 48S pre-initiation and 80S pretranslocation complexes. In the complex where the Sec-tRNA Sec was accommodated to the A site but transpeptidation was blocked, SBP2 bound the ribosome and possibly the SECIS element as well, and the SECIS had flexible contacts with the 60S ribosomal subunit involving several ribosomal proteins. Altogether, our findings led to broadening our understanding about the unique mechanism of selenocysteine incorporation in mammals.

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. Comparative analysis of peptides resulting from trypsinolysis of the crosslinked proteins before and after crosslink reversal reveals crosslinked peptides in the N-terminal parts of rpS5e and eIF2α. Application of these data to a model PIC structure obtained with the use of available structures indicates that eIF2α undergoes major conformation rearrangements to enable contacts of the factor with rpS5e. These contacts are suggested to maintain the correct positioning of eIF2α relative to other PIC components; this could be essential for start-codon selection by the PIC.

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