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J. Biol. Chem., Vol. 276, Issue 2, 1051-1056, January 12, 2001

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Biochemical Analysis of the eIF2 Complex Reveals a Structural Function for eIF2 in Catalyzed Nucleotide Exchange^*

Joseph Nika, Scott Rippel, and Ernest M. Hannig

From the Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75083

Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and delivers Met-tRNA_i^Met to the 40 S ribosomal subunit in a GTP-dependent manner. Initiation requires hydrolysis of eIF2-bound GTP, which releases an eIF2·GDP complex that is recycled to the GTP form by the nucleotide exchange factor eIF2B. The -subunit of eIF2 plays a critical role in regulating nucleotide exchange via phosphorylation at serine 51, which converts eIF2 into a competitive inhibitor of the eIF2B-catalyzed exchange reaction. We purified a form of eIF2 (eIF2) completely devoid of the -subunit to further study the role of eIF2 in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2 structural gene (SUI2). Removal of the -subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRNA_i^Met ligands by eIF2 in vitro. Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2 and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the -subunit increased K_m for eIF2·GDP by an order of magnitude, with a smaller increase in V_max. These data indicate that eIF2 is required for structural interactions between eIF2 and eIF2B that promote wild-type rates of nucleotide exchange. We suggest that this function contributes to the ability of the -subunit to control the rate of nucleotide exchange through reversible phosphorylation.

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