Fluorescence polarization studies of the interaction of Escherichia coli protein synthesis initiation factor 3 with 30S ribosomal subunits.
Steady-state fluorescence polarization techniques were used to study the binding of initiation factor 3 (IF3) to 30S ribosomal subunits. Covalent fluorescent derivatives of IF3 were prepared by treating the pure protein with fluorescein isothiocyanate. The fluorescein-labeled IF3 (F-IF3) contained 0.8--1.7 dye molecules per protein. Polyacrylamide gel electrophoretic analysis of the derivatized forms is consistent with the view that the probe is randomly attached, presumably to lysine epsilon-amino groups. The activity of F-IF3 is not impaired in assays for binding to 30S ribosomal subunits or in promoting formylmethionyl-tRNA binding to 70S ribosomes. Fluorescence polarization values were measured at different F-IF3 and 30S ribosomal subunit concentrations, and the association constant and number of binding sites were calculated. In buffer containing 10 mM magnesium acetate and 100 mM ammonium chloride, the association constant is (3.1 +/- 1.4) x 10(7) M-1, and the number of ribosomal binding sites is 1.2 +/- 0.2. The value for the association constant varies inversely with the ammonium chloride and magnesium acetate concentrations by a small amount. Competition studies show that nonderivatized IF3 binds to 30S ribosomal subunits with the same affinity as F-IF3. Therefore, the association constants measured for F-IF3 are valid for IF3 as well.
D000643:Ammonium Chloride / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005454:Fluorescence Polarization; D008274:Magnesium / Q000494:pharmacology; D010448:Peptide Initiation Factors / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3; D012270:Ribosomes / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D000643:Ammonium Chloride; D008274:Magnesium