Translation Initiation Fidelity Defective Mutations in eIF5 and eIF2β Show Distinct Sensitivity to the Sequence...

In Saccharomyces cerevisiae, the AUG codon typically signals translation initiation to set an open reading frame, with a preference for the A^- 3AA/U^- 1(AUG)U^+ 4 sequence context. The mutant eIF5^G31R or eIF2β^S264Y proteins cause translation initiation fidelity defect by initiating translation at a near cognate UUG codon, in addition to the AUG start codon (suppressor of initiation codon; Sui¯ phenotype). However, the critical role of the - 3 to - 1 sequences in selecting the UUG start codon by these Sui¯ mutants is not fully explored. Different HIS4^UUG-LacZ reporter constructs were made with UUG as the start codon and varied nucleotides at the - 3 and - 2 positions or individually at -1 positions. These constructs were transformed to yeast cells having eIF5^G31R or eIF2β^S264Y mutation, and the β-galactosidase activity was measured. The HIS4^UUG-LacZ transcripts carrying a purine (A/G) at the - 3 position showed higher reporter activity than those with a pyrimidine (U/C). Additionally, purines were favored at the - 1 position within an AA (- 3 and - 2) context for efficient UUG start codon selection. Our findings demonstrate that UUG start codon recognition by Sui¯ mutants eIF5^G31R and eIF2β^S264Y is greatly influenced by the surrounding nucleotide context, each showing distinct context preferences. This highlights the nuanced role of sequence context in the near-cognate UUG codon initiation by the Sui¯ mutants.