Wheat germ translation initiation factor eIF4B affects eIF4A and eIFiso4F helicase activity by increasing the ATP binding affinity of eIF4A.
It has been proposed that, during translational initiation, structures in the 5' untranslated region of mRNA are unwound. eIF4A, a member of the DEAD box family of proteins (those that contain a DEAD amino acid sequence), separately or in conjunction with other eukaryotic initiation factors, utilizes the energy from ATP hydrolysis to unwind these structures. As a step in defining the mechanism of helicase activity in the wheat germ protein synthesis system, we have utilized direct fluorescence measurements, ATPase assays, and helicase assays. The RNA duplex unwinding activity of wheat germ eIF4A is similar to other mammalian systems; however, eIF4F or eIFiso4F is required, probably because of the low binding affinity of wheat germ eIF4A for mRNA. Direct ATP binding measurements showed that eIF4A had a higher binding affinity for ADP than ATP, resulting in a limited hydrolysis and procession along the RNA in the helicase assay. The addition of eIF4B resulted in a change in binding affinity for ATP, increasing it almost 10-fold while the ADP binding affinity was approximately the same. The data presented in this paper suggest that eIF4F or eIFiso4F acts to position the eIF4A and stabilize the interaction with mRNA. ATP produces a conformational change which allows a limited unwinding of the RNA duplex. The binding of eIF4B either prior to or after hydrolysis allows for increased affinity for ATP and for the cycle of conformational changes to proceed, resulting in further unwinding and processive movement along the mRNA.
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Department of Chemistry, Hunter College and the Graduate Center, City University of New York, 695 Park Avenue, New York, New York 10021, USA.
Ren
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Goss
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D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D039601:Eukaryotic Initiation Factor-4A; D039642:Eukaryotic Initiation Factors; D005456:Fluorescent Dyes / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012315:RNA Caps / Q000138:chemical synthesis / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D017186:Titrimetry; D014908:Triticum
D039642:Eukaryotic Initiation Factors; D005456:Fluorescent Dyes; D010448:Peptide Initiation Factors; D012315:RNA Caps; C031799:eIF-4B; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D000255:Adenosine Triphosphate; D039601:Eukaryotic Initiation Factor-4A; D000251:Adenosine Triphosphatases; D020365:RNA Helicases; D008274:Magnesium