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A more recent version of this article appeared on January 5, 2001
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Papers In Press, published online ahead of print October 20, 2000
J. Biol. Chem, 10.1074/jbc.M007398200
Submitted on August 14, 2000
Revised on October 5, 2000
Accepted on October 20, 2000

Biochemical analysis of the eIF2beta gamma complex reveals a structural function for eIF2alpha in catalyzed nucleotide exchange

Joseph Nika, Scott Rippel, and Ernest M. Hannig

Molecular and Cell Biology, The University of Texas at Dallas, Richardson, TX 75083-0688

Corresponding Author: hannig{at}utdallas.edu

Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and delivers Met-tRNAiMet to the 40S 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 alpha -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 (eIF2beta gamma ) completely devoid of the alpha -subunit in order to further study the role of eIF2alpha in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2alpha structural gene (SUI2). Removal of the alpha -subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRNAiMet ligands by eIF2 in vitro. Qualitative assays to detect 43S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2beta gamma and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the alpha -subunit increased Km for eIF2beta gamma /GDP by an order of magnitude, with a smaller increase in Vmax. These data indicate that eIF2alpha 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 alpha -subunit to control the rate of nucleotide exchange through reversible phosphorylation.


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