Fluorescence study of the binding of m7GpppG and rabbit globin mRNA to protein synthesis initiation factors 4A, 4E, and 4F.
The interactions of protein synthesis initiation factors eIF-4E from human erythrocytes and eIF-4A and eIF-4F from rabbit reticulocytes with the cap analogue m7GpppG and rabbit globin mRNA were investigated. The equilibrium binding constants for the binary complex formation of eIF-4E-eIF-4A, m7GpppG-eIF-4E, m7GpppG-eIF-4F, globin mRNA-eIF-4E, globin mRNA-eIF-4F, and globin mRNA-eIF-4A were measured by direct fluorescence titration experiments. The binding of eIF-4E to globin mRNA was found to be 5.5-fold tighter than its binding to m7GpppG; the binding of eIF-4F for globin mRNA and m7GpppG was similar to that of eIF-4E. Association equilibrium constants were determined for the ternary system mRNA-eIF-4E-eIF-4A; four thermodynamically independent equilibria characterize the system. These equilibrium binding constants were used to calculate coupling free energies, which provided an estimate of the cooperativity of the interaction of eIF-4E, eIF-4A, and mRNA. These coupling energies were all found to be small and positive, indicative of anticooperative binding.
Goss
D J
DJ
Department of Chemistry, Hunter College City University of New York, New York 10021.
Carberry
S E
SE
Dever
T E
TE
Merrick
W C
WC
Rhoads
R E
RE
D001665:Binding Sites; D004912:Erythrocytes / Q000378:metabolism*; D039601:Eukaryotic Initiation Factor-4A; D039561:Eukaryotic Initiation Factor-4E; D039562:Eukaryotic Initiation Factor-4F; D005914:Globins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010448:Peptide Initiation Factors / Q000378:metabolism*; D012314:RNA Cap Analogs / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D039561:Eukaryotic Initiation Factor-4E; D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors; D012314:RNA Cap Analogs; D012315:RNA Caps; D012333:RNA, Messenger; C017516:7-methylguanosine triphosphate; D005914:Globins; D039601:Eukaryotic Initiation Factor-4A