Yeast Phenotypic Assays on Translational Control

This chapter describes phenotypic assays on specific and general aspects of translation using yeast Saccharomyces cerevisiae as a model eukaryote. To study the effect on start codon selection stringency, a his4^- or his4-lacZ allele altering the first AUG to AUU is employed. Mutations relaxing the stringent selection confer the His⁺ phenotype in the his4^- strain background or increase expression from his4-lacZ compared to that from wild-type HIS4-lacZ (Sui^- phenotype). Translation of the Gcn4p transcription activator is strictly regulated by amino acid availability depending on upstream ORF (uORF) elements in the GCN4 mRNA leader. Mutations reducing the eIF2/GTP/Met-tRNA_i ^Met complex level or the rate of its binding to the 40S subunit derepress GCN4 translation by allowing ribosomes to bypass inhibitory uORFs in the absence of the starvation signal (Gcd^- phenotype). Mutations impairing scanning or AUG recognition generally impair translational GCN4 induction during amino acid starvation (Gcn^- phenotype). Different amino acid analogs or amino acid enzyme inhibitors are used to study Gcd^- or Gcn^- phenotypes. The method of polysome profiling is also described to gain an ultimate "phenotypic" proof for translation defects.