Characterization of the interaction of wheat germ protein synthesis initiation factor eIF-3 with mRNA oligonucleotide and cap analogues.
Direct fluorescence titration experiments of wheat germ protein synthesis initiation factor eIF-3 with mRNA cap and oligoribonucleotide analogues were performed in order to determine the equilibrium association constants (Keq) for the eIF-3.mRNA interaction as a function of pH and temperature. These data suggest that (i) the eIF-3.mRNA interaction is not cap-specific (i.e., m7G-specific), (ii) ATP hydrolysis is not involved in the interaction, and (iii) the interaction is primarily ionic in nature. Competition experiments between a rabbit alpha-globin mRNA oligoribonucleotide analogue and either mRNA cap analogues or nucleoside triphosphates (NTPs) are also reported; these experiments indicate that NTPs act as both activators and competitive inhibitors of the mRNA.eIF-3 association. The results are consistent with a partially uncompetitive binding mechanism, whereby at low NTP concentrations (less than or equal to 10 microM) the bound NTP enhances subsequent mRNA binding to eIF-3, perhaps by inducing a conformational change, and at higher NTP concentrations, the NTP acts as a competitive inhibitor for the mRNA binding site on eIF-3.
Carberry
S E
SE
Department of Chemistry, Hunter College, City University of New York, New York 10021-5024.
Goss
D J
DJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D039621:Eukaryotic Initiation Factor-3; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics*; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000302:isolation & purification; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification; D012315:RNA Caps / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014908:Triticum / Q000737:chemistry*
D039621:Eukaryotic Initiation Factor-3; D009841:Oligonucleotides; D010448:Peptide Initiation Factors; D010940:Plant Proteins; D012315:RNA Caps; D000255:Adenosine Triphosphate