The DEAD box protein eIF4A. 1. A minimal kinetic and thermodynamic framework reveals coupled binding of RNA and nucleotide.
eIF4A is the archetypal member of the DEAD box family of proteins and has been proposed to use the energy from ATP hydrolysis to unwind structures in the 5'-untranslated regions of eukaryotic mRNAs during translation initiation. As a step toward understanding the mechanism of action of this class of enzymes, a minimal kinetic and thermodynamic framework for the RNA-activated ATPase function has been established for eIF4A. The enzyme's affinity for ssRNA is modulated by the binding of ATP.Mg2+ and ADP.Mg2+: the affinity of the E.ATP complex for ssRNA is approximately 40-fold higher than that of the E.ADP complex. The enzyme binds its substrates in a random manner; contrary to previous suggestions, neither ATP binding nor hydrolysis is required for binding of single-stranded RNA. The presence or absence of the gamma-phosphate on the bound nucleotide acts as a switch that modulates the enzyme's structure and ssRNA affinity. The data presented in this and the following paper in this issue suggest that ATP binding and hydrolysis produce a cycle of conformational and RNA affinity changes in eIF4A. Such cycles may be used by DEAD box proteins to transduce the energy from ATP hydrolysis into physical work, thereby allowing each member of this family to rearrange its RNA or RNA.protein target.
Lorsch
J R
JR
Department of Biochemistry, Beckman Center, B400, Stanford University, Stanford, California 94305-5307, USA.
Herschlag
D
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D000227:Adenine Nucleotides / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D039601:Eukaryotic Initiation Factor-4A; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation / Q000528:radiation effects; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014466:Ultraviolet Rays
D000227:Adenine Nucleotides; D003432:Cross-Linking Reagents; D010448:Peptide Initiation Factors; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; D012313:RNA; D039601:Eukaryotic Initiation Factor-4A; D012316:RNA Nucleotidyltransferases