Eukaryotic Translation Initiation Factor 5 Is Critical for Integrity of the Scanning Preinitiation Complex and Accurate Control of GCN4 Translation

Abstract

The integrity of eukaryotic translation initiation factor (eIF) interactions in ribosomal preinitiation complexes is critical for the proper regulation of GCN4 mRNA translation in response to amino acid availability. Increased phosphorylation of eIF2 under amino acid starvation conditions leads to a corresponding increase in GCN4 mRNA translation. The carboxyl-terminal domain (CTD) of eIF5 (eIF5-CTD) has been identified as a potential nucleation site for preinitiation complex assembly. To further characterize eIF5 and delineate its role in GCN4 translational control, we isolated mutations leading to temperature sensitivity (Ts⁻ phenotype) targeted at TIF5, the structural gene encoding eIF5 in yeast (Saccharomyces cerevisiae). Nine single point mutations were isolated, in addition to an allele in which the last 15 amino acids were deleted. The nine point mutations clustered in the eIF5-CTD, which contains two conserved aromatic/acidic boxes. Six of the point mutations derepressed GCN4 translation independent of eIF2 phosphorylation (Gcd⁻ phenotype) at a permissive temperature, directly implicating eIF5-CTD in the eIF2/GTP/Met-tRNA_i^Met ternary complex binding process required for GCN4 translational control. In addition, stronger restriction of eIF5-CTD function at an elevated temperature led to failure to derepress GCN4 translation (Gcn⁻ phenotype) in all of the mutants, most likely due to leaky scanning of the first upstream open reading frame of GCN4 mRNA. This latter result directly implicates eIF5-CTD in the process of accurate scanning for, or recognition of, AUG codons. Taken together, our results indicate that eIF5-CTD plays a critical role in both the assembly of the 43S complex and the postassembly process in the 48S complex, likely during the scanning process.

ACKNOWLEDGMENTS

We are indebted to Alan Hinnebusch for timely advice, the gift of plasmids, and critical comments on the manuscript. We also thank Ashik Srinivasan for initial observation of Gcd⁻ phenotypes caused by some eIF5-CTD mutants, Beth Montelone for critical reading of the manuscript, and Assen Marintchev for inspiring discussion about the structure of eIF5-CTD.

This work was supported by the NIH COBRE awards P20 RR15563 and RR16475, matching support from the State of Kansas and the KSU, and NIH grant R01GM64781 to K.A. and ACS grant RPG-97-061-01-NP to E.M.H.