Translation Initiation Factor 2γ Mutant Alters Start Codon Selection Independent of Met-tRNA Binding

Abstract

Selection of the AUG start codon for translation in eukaryotes is governed by codon-anticodon interactions between the initiator Met-tRNA_i^Met and the mRNA. Translation initiation factor 2 (eIF2) binds Met-tRNA_i^Met to the 40S ribosomal subunit, and previous studies identified Sui⁻ mutations in eIF2 that enhanced initiation from a noncanonical UUG codon, presumably by impairing Met-tRNA_i^Met binding. Consistently, an eIF2γ-N135D GTP-binding domain mutation impairs Met-tRNA_i^Met binding and causes a Sui⁻ phenotype. Intragenic A208V and A382V suppressor mutations restore Met-tRNA_i^Met binding affinity and cell growth; however, only A208V suppresses the Sui⁻ phenotype associated with the eIF2γ-N135D mutation. An eIF2γ-A219T mutation impairs Met-tRNA_i^Met binding but unexpectedly enhances the fidelity of initiation, suppressing the Sui⁻ phenotype associated with the eIF2γ-N135D,A382V mutant. Overexpression of eIF1, which is thought to monitor codon-anticodon interactions during translation initiation, likewise suppresses the Sui⁻ phenotype of the eIF2γ mutants. We propose that structural alterations in eIF2γ subtly alter the conformation of Met-tRNA_i^Met on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNA_i^Met binding affinity.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank Alan Hinnebusch and Jon Lorsch for comments on the manuscript and helpful discussions; Lee Kapp, Mike Acker, Mikkel Algire, and Jon Lorsch for help in establishing the biochemical assays; members of the Hinnebusch and Dever labs for helpful suggestions; Tom Donahue for plasmids; and John Hershey for support.

This work was supported in part by the Intramural Program of the NIH, NICHD (T.E.D.).