Kinetic mechanism for assembly of the m7GpppG.eIF4E.eIF4G complex.
Interaction of the mRNA cap with the translational machinery is a critical and early step in the initiation of protein synthesis. To better understand this process, we determined kinetic constants for the interaction of m(7)GpppG with human eIF4E by stopped-flow fluorescence quenching in the presence of a 90-amino acid fragment of human eIF4G that contains the eIF4E-binding domain (eIF4G(557-646)). The values obtained, k(on) = 179 x 10(6) m(-1) s(-1) and k(off) = 79 s(-1), were the same as reported previously in the absence of an eIF4G-derived peptide. We also used surface plasmon resonance to determine kinetic constants for the binding of eIF4E to eIF4G(557-646), both in the presence and absence of m(7)GpppG. The results indicated that eIF4G(557-646) binds eIF4E and eIF4E.m(7)GpppG at the same rate, with k(on) = 3 x 10(6) m(-1) s(-1) and k(off) = 0.01 s(-1). Our data represent the first full kinetic description of the interaction of eIF4E with its two specific ligands. The results demonstrate that the formation of the m(7)GpppG.eIF4E.eIF4G(557-646) complex obeys a sequential, random kinetic mechanism and that there is no preferential pathway for its formation. Thus, even though eIF4G(557-646) binds eIF4E tightly, it does not increase the affinity of eIF4E for m(7)GpppG, as has been claimed in several previous publications. We did, in fact, observe increased binding to m(7)GTP-Sepharose in the presence of eIF4G(557-646), but only with recombinant eIF4E that was prepared from inclusion bodies.
Slepenkov
Sergey V
SV
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
Korneeva
Nadejda L
NL
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932.
Rhoads
Robert E
RE
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932. Electronic address: rrhoad@lsuhsc.edu.
D000595:Amino Acid Sequence; D039561:Eukaryotic Initiation Factor-4E / Q000737:chemistry* / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012314:RNA Cap Analogs / Q000737:chemistry*; D012315:RNA Caps / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D008024:Ligands; D012314:RNA Cap Analogs; D012315:RNA Caps; D012685:Sepharose