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.
Lee
Bumjun
B
Molecular, Cellular, and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas, USA.
Udagawa
Tsuyoshi
T
Singh
Chingakham Ranjit
CR
Asano
Katsura
K
D050976:Basic-Leucine Zipper Transcription Factors; D002499:Centrifugation, Density Gradient; D018387:Codon, Initiator; D004268:DNA-Binding Proteins / Q000502:physiology; D005822:Genetic Vectors; D010442:Peptide Chain Initiation, Translational / Q000502:physiology*; D010641:Phenotype; D011132:Polyribosomes / Q000502:physiology; D014176:Protein Biosynthesis / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology; D014157:Transcription Factors / Q000502:physiology; D014170:Transformation, Genetic
D050976:Basic-Leucine Zipper Transcription Factors; D018387:Codon, Initiator; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors