Abstract
The small ribosomal subunit protein uS9 (formerly called rpS16 in Saccharomyces cerevisiae), has a long protruding C-terminal tail (CTT) that extends towards the mRNA cleft of the ribosome. The last C-terminal residue of uS9 is an invariably conserved, positively charged Arg that is believed to enhance interaction of the negatively charged initiator tRNA with the ribosome when the tRNA is base-paired to the AUG codon in the P-site. In order to more fully characterize the role of the uS9 CTT in eukaryotic translation, we tested how truncations, extensions and substitutions within the CTT affect initiation and elongation processes in Saccharomyces cerevisiae. We found that uS9 C-terminal residues are critical for efficient recruitment of the eIF2•GTP•Met-tRNAiMet ternary complex to the ribosome and for its proper response to the presence of an AUG codon in the P-site during the scanning phase of initiation. These residues also regulate hydrolysis of the GTP in the eIF2•GTP•Met-tRNAiMet complex to GDP and Pi. In addition, our data show that uS9 CTT modulates elongation fidelity. Therefore, we propose that uS9 CTT is critical for proper control of the complex interplay of events surrounding accommodation of initiator and elongator tRNAs in the P- and A-sites of the ribosome.
Highlights
In both bacterial and eukaryotic systems, protein synthesis consists of four major steps: initiation, elongation, termination and ribosome recycling
We previously demonstrated that yeast uS9 mutants lacking the last two C-terminal residues of the C-terminal tail (CTT) or containing a neutral Gly in place of the last C-terminal Arg of the CTT displayed a slow-growth (Slg−) phenotype, reduced rate of bulk translation initiation, impaired derepression of GCN4 mRNA translation (Gcn− phenotype) and accumulation of eIF1 and eukaryotic initiation factor 2 (eIF2) on native 40S subunits [7]
We proposed that the uS9 CTT influences initiation events surrounding recruitment of the eIF2GTPMet-tRNAiMet ternary complex (TC) and promotes eIF5-stimulated GTPhydrolysis or Pi release [7]
Summary
In both bacterial and eukaryotic systems, protein synthesis (translation) consists of four major steps: initiation, elongation, termination and ribosome recycling (for review, see [1,2,3,4]). The elongation phase involves sequential addition of the subsequent amino acids encoded by the mRNA to the nascent polypeptide chain This phase is critical for maintaining the accuracy of protein synthesis as elongator aminoacyl (aa)-tRNAs must correctly ‘translate’ mRNA codons by bringing the correct amino acid to the ribosome. Recognition of the start codon leads to displacement of eIF1, followed by deep insertion of the anticodon stem loop (ASL) of Met-tRNAiMet into the P-site (PIN state). This results in a closed conformation (characteristic for the 48S complex) and eIF5-stimulated GTP hydrolysis and Pi
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