Kinetic mechanism for the binding of eIF4F and tobacco Etch virus internal ribosome entry site rna: effects of eIF4B and poly(A)-binding protein.
The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (k(on)) and dissociation rate constant (k(off)) for eIF4F binding to IRES were 59 +/- 2.1 micro s(-1) and 12.9 +/- 0.3 s(-1), respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F approximately 3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES.
Department of Chemistry and Biochemistry, Hunter College and Graduate Center of the City University of New York, New York, New York 10065, USA.
Mateen A
MA
Khan
Hasan
H
Yumak
Dixie J
DJ
Goss
D039562:Eukaryotic Initiation Factor-4F / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010442:Peptide Chain Initiation, Translational / Q000502:physiology*; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D017800:Potyvirus / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D012315:RNA Caps / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D014908:Triticum / Q000737:chemistry* / Q000378:metabolism
D039562:Eukaryotic Initiation Factor-4F; D010940:Plant Proteins; D012315:RNA Caps; D012367:RNA, Viral