RESUMO
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.
Assuntos
Códon de Terminação/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Terminação Traducional da Cadeia Peptídica , Proteínas Ribossômicas/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Sítios de Ligação/genética , Fator de Iniciação 3 em Eucariotos/genética , Organismos Geneticamente Modificados , Terminação Traducional da Cadeia Peptídica/genética , Ligação Proteica , Biossíntese de Proteínas/genética , RNA de Transferência/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans.
