| Details: |
In eukaryotes the translation initiation factor eIF2 plays a key role in the selection of the start codon. The GTP-bound eIF2 binds Met-tRNAiMet forming a ternary complex (TC) and then delivers the Met-tRNAiMet to the 40S ribosomal subunit. The AUG codon selection is governed by codon-anticodon interaction between the mRNA and Met-tRNAiMet and by GTP hydrolysis and Pi release by eIF2. Previous studies identified Sui- mutations in eIF2 that enhanced initiation from a non-canonical UUG codon by weakening Met-tRNAiMet binding. Consistently, an eIF2g-N135D mutation in the Switch I element impaired Met-tRNAiMet binding both in vitro and in vivo without affecting eIF2 and eIF2 binding and caused both a slow-growth and a Sui- phenotype in yeast. Intragenic A208V (switch II) and A382V (domain II) suppressor mutations were identified that restored yeast cell growth. These suppressor mutations restored Met-tRNAiMet binding both in vitro and in vivo, however, only the switch II A208V mutation suppressed the Sui- phenotype associated with the eIF2g-N135D mutation. An eIF2g-A219T mutation located at the base of the GTP-binding pocket in the G-domain impaired Met-tRNAiMet binding but unexpectedly enhanced the fidelity of initiation, suppressing the Sui- phenotype associated with the eIF2g-N135D,A382V mutation.
Overexpression of eIF1, which is thought to monitor codon-anticodon interactions during translation initiation, likewise suppressed the Sui- phenotype of the eIF2g mutants. These new mutations in eIF2 reveal an alternate mechanism for Sui- phenotypes. Accordingly, the mutations introduce structural alterations in eIF2g that alter the conformation of Met-tRNAiMet on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNAiMet binding affinity.
|