Depletion and deletion analyses of eucaryotic translation initiation factor 1A in Saccharomyces cerevisiae

Translation initiation factor eIF1A is a highly conserved, small, acidic pr otein that is required for cell growth in yeast. Biochemical studies in vit ro implicate eIF1A in dissociating ribosomes, promoting methionyl-tRNA(i) b inding 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 pr omoter. After Saccharomyces cerevisiae cells were shifted to glucose medium , depletion of eIF1A was seen after 3-4 generations, corresponding with ces sation of cell growth. Polysome profiles of the depleted strain showed ribo some run-off from mRNAs, indicating that eIF1A is involved in the initiatio n phase of translation. A decrease in free 40S ribosomes and an apparent in crease in free 60S ribosomes were attributed to the formation of 40S subuni t dimers. The result suggests that one of the functions of eIF1A is to prev ent formation of 40S dimers. Mutant forms of CIF I A lacking either the pos itively charged N-terminal region or the negatively charged C-terminal regi on were constructed and tested for their ability to confer cell growth as t he sole source of eIF1A. Either deletion supports cell growth, albeit at a slower rate, and causes a reduction in polysomes, although eIF1A lacking th e N-terminal region is more deleterious. Therefore the charged terminal reg ions contribute to, but are not absolutely essential for, eIF1A function. ( C) 2001 Societe francaise de biochimie et biologic moleculaire / Editions s cientifiques et medicales Elsevier SAS. All rights reserved.