The 40S ribosome plays a critical role in start codon selection. To gain insights into the role of its 18S rRNA in start codon selection, a suppressor screen was performed that suppressed the preferential UUG start codon recognition (Suppressor of initiation codon: Sui− phenotype) associated with the eIF5G31R mutant. The C1209U mutation in helix h32 of 18S rRNA was found to suppress the Sui− and Gcn− (failure to derepress GCN4 expression) phenotype of the eIF5G31R mutant. The C1209U mutation suppressed Sui− and Gcd− (constitutive derepression of GCN4 expression) phenotype of eIF2βS264Y, eIF1K60E, and eIF1A‐ΔC mutation. We propose that the C1209U mutation in 40S ribosomal may perturb the premature head rotation in ‘Closed/PIN’ state and enhance the stringency of translation start site selection.

The recognition of the AUG start codon and selection of an open reading frame (ORF) is fundamental to protein biosynthesis. Defect in the fidelity of start codon selection adversely affect proteome and have a pleiotropic effect on cellular function. Using proteomic techniques, we identified differential protein abundance in the translation initiation fidelity defective eIF5G31R mutant that initiates translation using UUG codon in addition to the AUG start codon. Consistently, the eIF5G31R mutant altered proteome involved in protein catabolism, nucleotide biosynthesis, lipid biosynthesis, carbohydrate metabolism, oxidation–reduction pathway, autophagy and re-programs the cellular pathways. The utilization of the upstream UUG codons by the eIF5G31R mutation caused downregulation of uridylate kinase expression, sensitivity to hydroxyurea, and DNA damage. The eIF5G31R mutant cells showed lower glutathione levels, high ROS activity, and sensitivity to H2O2.

In higher eukaryotes, the efficiency of start codon selection depends upon its sequence context A/GxxAUGG, especially at the -3 and +4 position. However, S. cerevisiae prefers AAA/UAUGU sequence context surrounding the AUG start codon. Furthermore, it was demonstrated that the first AUG codon on the mRNA serves as a signal for ribosomal recognition of the initiation site selection. In yeast, the G31R mutation in eIF5 shows Suppressor of initiation codon (Sui) phenotype enabling 40S ribosome to initiate at UUG start codon. To understand whether the UUG codon recognition by the eIF5G31R mutant is sensitive to the sequence context, we made different HIS4-LacZ reporter constructs with UUG as start codon and changed the sequence context at -3 and -2 positions. The HIS4UUG-LacZ transcripts that carry purine (A/G) at the -3-position showed better β-Galactosidase activity than pyrimidine (U/C). Furthermore, on changing the -1 position for the AAA context, we discovered purines are more preferred at the -1 position for the UUG start codon selection.