Homeostasis in mRNA initiation: wheat germ poly(A)-binding protein lowers the activation energy barrier to initiation complex formation.
Previous kinetic binding studies of wheat germ protein synthesis eukaryotic initiation factor iso4F (eIFiso4F) and its subunit, eIF4E, with m(7)GTP and mRNA analogues indicated that binding occurred by a two-step process with the first step being too fast to measure by stopped-flow techniques (). Further equilibrium studies showed that poly(A)-binding protein (PABP) enhanced the cap binding of eIFiso4F about 40-fold. The kinetic effects of PABP on cap binding and the temperature dependence of this reaction were measured and compared. Fluorescence stopped-flow studies of the PABP.eIFiso4F protein complex with cap show a concentration-independent conformational change. PABP did not significantly increase the rate of the conformational change, and because the initial second-order binding is essentially diffusion-controlled, the enhancement of cap affinity must reside in the dissociation rate. The dissociation rate was more than 5-fold slower in the presence of PABP. The temperature dependence of the cap binding reaction was markedly reduced in the presence of PABP. The reduced energy barrier for formation of a cap.eIFiso4F complex suggests a more stable platform for further initiation complex formation and a possible means of adapting to varying temperature conditions.
Luo
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Department of Chemistry, Hunter College and the Graduate Center of the City University of New York, New York, New York 10021, USA.
Goss
D J
DJ
D039561:Eukaryotic Initiation Factor-4E; D039562:Eukaryotic Initiation Factor-4F; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D010448:Peptide Initiation Factors / Q000378:metabolism; D039102:Poly(A)-Binding Proteins; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D014908:Triticum / Q000378:metabolism*
D039561:Eukaryotic Initiation Factor-4E; D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors; D039102:Poly(A)-Binding Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D006160:Guanosine Triphosphate