Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.

• MeSH
• Eukaryotic Initiation Factor-3 genetics   metabolism   MeSH
• Organisms, Genetically Modified MeSH
• Protein Biosynthesis genetics   MeSH
• Ribosomal Proteins genetics   physiology   MeSH
• Ribosomes metabolism   MeSH
• RNA, Transfer metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   physiology   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Peptide Chain Termination, Translational * genetics   MeSH
• Codon, Terminator metabolism   MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• Ribosomal Proteins MeSH
• RNA, Transfer MeSH
• RPS3 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Codon, Terminator MeSH

eIF3 is a large multiprotein complex serving as an essential scaffold promoting binding of other eIFs to the 40S subunit, where it coordinates their actions during translation initiation. Perhaps due to a high degree of flexibility of multiple eIF3 subunits, a high-resolution structure of free eIF3 from any organism has never been solved. Employing genetics and biochemistry, we previously built a 2D interaction map of all five yeast eIF3 subunits. Here we further improved the previously reported in vitro reconstitution protocol of yeast eIF3, which we cross-linked and trypsin-digested to determine its overall shape in 3D by advanced mass-spectrometry. The obtained cross-links support our 2D subunit interaction map and reveal that eIF3 is tightly packed with its WD40 and RRM domains exposed. This contrasts with reported cryo-EM structures depicting eIF3 as a molecular embracer of the 40S subunit. Since the binding of eIF1 and eIF5 further fortified the compact architecture of eIF3, we suggest that its initial contact with the 40S solvent-exposed side makes eIF3 to open up and wrap around the 40S head with its extended arms. In addition, we mapped the position of eIF5 to the region below the P- and E-sites of the 40S subunit.

• MeSH
• Cryoelectron Microscopy MeSH
• Eukaryotic Initiation Factor-1 chemistry   genetics   metabolism   MeSH
• Eukaryotic Initiation Factor-3 chemistry   genetics   metabolism   MeSH
• Eukaryotic Initiation Factor-5 chemistry   genetics   metabolism   MeSH
• Peptide Chain Initiation, Translational * MeSH
• Ribosome Subunits, Small, Eukaryotic genetics   metabolism   MeSH
• Models, Molecular MeSH
• Protein Domains MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   ultrastructure   MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Eukaryotic Initiation Factor-1 MeSH
• Eukaryotic Initiation Factor-3 MeSH
• Eukaryotic Initiation Factor-5 MeSH
• Saccharomyces cerevisiae Proteins MeSH

Protein production must be strictly controlled at its beginning and end to synthesize a polypeptide that faithfully copies genetic information carried in the encoding mRNA. In contrast to viruses and prokaryotes, the majority of mRNAs in eukaryotes contain only one coding sequence, resulting in production of a single protein. There are, however, many exceptional mRNAs that either carry short open reading frames upstream of the main coding sequence (uORFs) or even contain multiple long ORFs. A wide variety of mechanisms have evolved in microbes and higher eukaryotes to prevent recycling of some or all translational components upon termination of the first translated ORF in such mRNAs and thereby enable subsequent translation of the next uORF or downstream coding sequence. These specialized reinitiation mechanisms are often regulated to couple translation of the downstream ORF to various stimuli. Here we review all known instances of both short uORF-mediated and long ORF-mediated reinitiation and present our current understanding of the underlying molecular mechanisms of these intriguing modes of translational control.

• MeSH
• Bacteria genetics   metabolism   MeSH
• Eukaryota genetics   MeSH
• Humans MeSH
• Open Reading Frames genetics   MeSH
• Protein Biosynthesis genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Intramural MeSH

Protein synthesis is mediated via numerous molecules including the ribosome, mRNA, tRNAs, as well as translation initiation, elongation and release factors. Some of these factors play several roles throughout the entire process to ensure proper assembly of the preinitiation complex on the right mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most intriguing of these multitasking factors is the eukaryotic initiation factor eIF3. Recent evidence strongly suggests that this factor, which coordinates the progress of most of the initiation steps, does not come off the initiation complex upon subunit joining, but instead it remains bound to 80S ribosomes and gradually falls off during the first few elongation cycles to: (1) promote resumption of scanning on the same mRNA molecule for reinitiation downstream-in case of translation of upstream ORFs short enough to preserve eIF3 bound; or (2) come back during termination on long ORFs to fine tune its fidelity or, if signaled, promote programmed stop codon readthrough. Here, we unite recent structural views of the eIF3-40S complex and discus all known eIF3 roles to provide a broad picture of the eIF3's impact on translational control in eukaryotic cells.

• MeSH
• Eukaryotic Initiation Factor-3 chemistry   genetics   metabolism   MeSH
• Protein Conformation * MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Models, Molecular MeSH
• Protein Subunits chemistry   genetics   metabolism   MeSH
• Protein Biosynthesis * MeSH
• Ribosomes genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• RNA, Messenger MeSH
• Protein Subunits MeSH
• Saccharomyces cerevisiae Proteins MeSH

Translation reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream ORFs to prevent recycling of the post-termination 40S subunit in order to resume scanning for reinitiation downstream. Its efficiency decreases with the increasing uORF length, or by the presence of secondary structures, suggesting that the time taken to translate a uORF is more critical than its length. This led to a hypothesis that some initiation factors needed for reinitiation are preserved on the 80S ribosome during early elongation. Here, using the GCN4 mRNA containing four short uORFs, we developed a novel in vivo RNA-protein Ni2+-pull down assay to demonstrate for the first time that one of these initiation factors is eIF3. eIF3 but not eIF2 preferentially associates with RNA segments encompassing two GCN4 reinitiation-permissive uORFs, uORF1 and uORF2, containing cis-acting 5΄ reinitiation-promoting elements (RPEs). We show that the preferred association of eIF3 with these uORFs is dependent on intact RPEs and the eIF3a/TIF32 subunit and sharply declines with the extended length of uORFs. Our data thus imply that eIF3 travels with early elongating ribosomes and that the RPEs interact with eIF3 in order to stabilize the mRNA-eIF3-40S post-termination complex to stimulate efficient reinitiation downstream.

• MeSH
• 5' Untranslated Regions MeSH
• Peptide Chain Elongation, Translational MeSH
• Eukaryotic Initiation Factor-3 metabolism   MeSH
• Genetic Techniques MeSH
• Peptide Chain Initiation, Translational * MeSH
• Ribosome Subunits, Small, Eukaryotic metabolism   MeSH
• Open Reading Frames * MeSH
• Gene Expression Regulation * MeSH
• Ribosomes metabolism   MeSH
• Peptide Chain Termination, Translational MeSH
• Codon, Terminator MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5' Untranslated Regions MeSH
• Eukaryotic Initiation Factor-3 MeSH
• Codon, Terminator MeSH