ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors.
Interaction of protein synthesis initiation factors with mRNA has been studied in order to characterize early events in the eukaryotic translation pathway. Individual reovirus mRNAs labeled with 32P in the alpha position relative to the m7G cap and eukaryotic initiation factor (eIF)-4A, -4B, and -4F purified from rabbit reticulocytes were employed. It was found that eIF-4A causes a structural change in mRNA, as evidenced by a nuclease sensitivity test: addition of high concentrations of eIF-4A greatly increase the nuclease sensitivity of the mRNA, suggesting that this factor can melt or "unwind" mRNA structure. ATP is required for this reaction. At low concentrations of eIF-4A, addition of eIF-4B is required for maximal unwinding activity. Thus eIF-4B enhances eIF-4A activity. Addition of eIF-4F also makes the mRNA sensitive to nuclease indicating a similar unwinding role to that of eIF-4A. Stoichiometric comparisons indicate that eIF-4F is more than 20-fold more efficient than eIF-4A in catalyzing this reaction. The unwinding activity of eIF-4F is inhibited by m7GDP, while that of eIF-4A is not. This suggests that eIF-4A functions independent of the 5' cap structure. Our results also suggest that the unwinding activity of eIF-4F is located in the 46,000-dalton polypeptide of this complex, which has shown by others to be similar or identical to eIF-4A.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004247:DNA / Q000378:metabolism; D039601:Eukaryotic Initiation Factor-4A; D039562:Eukaryotic Initiation Factor-4F; D039642:Eukaryotic Initiation Factors; D007739:L Cells / Q000378:metabolism; D012089:Mammalian orthoreovirus 3 / Q000235:genetics*; D051379:Mice; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D012315:RNA Caps / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D012087:Reoviridae / Q000235:genetics*; D012156:Reticulocytes / Q000378:metabolism
D039562:Eukaryotic Initiation Factor-4F; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; D010761:Phosphorus Radioisotopes; D012315:RNA Caps; D012333:RNA, Messenger; C031799:eIF-4B; D000255:Adenosine Triphosphate; D004247:DNA; D039601:Eukaryotic Initiation Factor-4A