Depletion and deletion analyses of eucaryotic translation initiation factor 1A in Saccharomyces cerevisiae.
Translation initiation factor eIF1A is a highly conserved, small, acidic protein that is required for cell growth in yeast. Biochemical studies in vitro implicate eIF1A in dissociating ribosomes, promoting methionyl-tRNA(i) binding to 40S ribosomal subunits, scanning of mRNAs and recognizing the AUG initiation codon. To elucidate the pleiotropic functions of eIF1A in vivo, the factor was depleted by placing its gene behind the repressible GAL1 promoter. After Saccharomyces cerevisiae cells were shifted to glucose medium, depletion of eIF1A was seen after 3-4 generations, corresponding with cessation of cell growth. Polysome profiles of the depleted strain showed ribosome run-off from mRNAs, indicating that eIF1A is involved in the initiation phase of translation. A decrease in free 40S ribosomes and an apparent increase in free 60S ribosomes were attributed to the formation of 40S subunit dimers. The result suggests that one of the functions of eIF1A is to prevent formation of 40S dimers. Mutant forms of eIF1A lacking either the positively charged N-terminal region or the negatively charged C-terminal region were constructed and tested for their ability to confer cell growth as the sole source of eIF1A. Either deletion supports cell growth, albeit at a slower rate, and causes a reduction in polysomes, although eIF1A lacking the N-terminal region is more deleterious. Therefore the charged terminal regions contribute to, but are not absolutely essential for, eIF1A function.
Kainuma
M
M
Department of Biological Chemistry, School of Medicine, University of California, Davis, CA 95616, USA.
Hershey
J W
JW
D000595:Amino Acid Sequence; D015153:Blotting, Western; D005057:Eukaryotic Cells; D015977:Eukaryotic Initiation Factor-1; D005656:Fungal Proteins / Q000096:biosynthesis; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000378:metabolism*; D011132:Polyribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics*; D013997:Time Factors
D015977:Eukaryotic Initiation Factor-1; D005656:Fungal Proteins; D010448:Peptide Initiation Factors; C026022:eukaryotic peptide initiation factor-1A