Transfer RNAs (tRNAs) serve as a dictionary for the ribosome translating the genetic message from mRNA into a polypeptide chain. In addition to this canonical role, tRNAs are involved in other processes such as programmed stop codon readthrough (SC-RT). There, tRNAs with near-cognate anticodons to stop codons must outcompete release factors and incorporate into the ribosomal decoding center to prevent termination and allow translation to continue. However, not all near-cognate tRNAs promote efficient SC-RT. Here, with the help of Saccharomyces cerevisiae and Trypanosoma brucei, we demonstrate that those tRNAs that promote efficient SC-RT establish critical contacts between their anticodon stem (AS) and ribosomal proteins Rps30/eS30 and Rps25/eS25 forming the decoding site. Unexpectedly, the length and well-defined nature of the AS determine the strength of these contacts, which is reflected in organisms with reassigned stop codons. These findings open an unexplored direction in tRNA biology that should facilitate the design of artificial tRNAs with specifically altered decoding abilities.

• MeSH
• Anticodon metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Protein Biosynthesis * MeSH
• Ribosomal Proteins metabolism   MeSH
• Ribosomes * metabolism   MeSH
• RNA, Transfer * metabolism   genetics   chemistry   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Codon, Terminator * genetics   metabolism   MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anticodon MeSH
• Ribosomal Proteins MeSH
• RNA, Transfer * MeSH
• Codon, Terminator * MeSH

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

Reinitiation after translation of short upstream ORFs (uORFs) represents one of the means of regulation of gene expression on the mRNA-specific level in response to changing environmental conditions. Over the years it has been shown-mainly in budding yeast-that its efficiency depends on cis-acting features occurring in sequences flanking reinitiation-permissive uORFs, the nature of their coding sequences, as well as protein factors acting in trans. We earlier demonstrated that the first two uORFs from the reinitiation-regulated yeast GCN4 mRNA leader carry specific structural elements in their 5' sequences that interact with the translation initiation factor eIF3 to prevent full ribosomal recycling post their translation. Actually, this interaction turned out to be instrumental in stabilizing the mRNA·40S post-termination complex, which is thus capable to eventually resume scanning and reinitiate on the next AUG start site downstream. Recently, we also provided important in vivo evidence strongly supporting the long-standing idea that to stimulate reinitiation, eIF3 has to remain bound to ribosomes elongating these uORFs until their stop codon has been reached. Here we examined the importance of eIF3 and sequences flanking uORF1 of the human functional homolog of yeast GCN4, ATF4, in stimulation of efficient reinitiation. We revealed that the molecular basis of the reinitiation mechanism is conserved between yeasts and humans.

• Keywords
• ATF4, GCN4, eIF3, mRNA, reinitiation, ribosome, translational control,
• MeSH
• Eukaryotic Initiation Factor-3 chemistry   metabolism   MeSH
• Peptide Chain Initiation, Translational * MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Open Reading Frames * MeSH
• Protein Biosynthesis MeSH
• Ribosomes metabolism   MeSH
• Mammals MeSH
• Activating Transcription Factor 4 chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• RNA, Messenger MeSH
• Activating Transcription Factor 4 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