The beta subunit of eukaryotic translation initiation factor 2 binds mRNA through the lysine repeats and a region comprising the C-2-C-2 motif

Eukaryotic translation initiation factor 2 (eIF2) has been implicated in th e selection of the AUG codon as the start site for eukaryotic translation i nitiation, since mutations in its three subunits in yeast that allow the re cognition of a UUG codon by the anticodon of the initiator Met-tRNA(Met) ha ve been identified. All such mutations in the beta subunit of eIF2 (eIF2 be ta) mapped to a region containing a putative zinc finger structure of the C -2-C-2 type, indicating that these sequences could be involved in RNA recog nition. Another feature of eIF2 beta that could mediate an interaction with RNA is located in the amino-terminal sequences and is composed of three re peats of seven lysine residues which are highly conserved in other species. We show here the ability of eIF2 beta, purified from Escherichia coli as a fusion to glutathione S-transferase, to bind mRNA in vitro. Through a dele tion analysis, mRNA binding was found to be dependent on the lysine repeats and a region encompassing the C-2-C-2 motif. Strong mRNA binding in vitro could be maintained by the presence of only one lysine or one arginine run but not one alanine run. We further show that only one run of lysine residu es is sufficient for the in vivo function of eIF2 beta, probably through ch arge interaction, since its replacement by arginines did not impair cell vi ability, whereas substitution for alanines resulted in inviable cells, mRNA binding, but not GTP-dependent initiator Met-tRNA(Met) binding, by the eIF 2 complex was determined to be dependent on the presence of the lysine runs of the beta subunit.