PMID: 7028112Sep 29, 1981Paper

Fluorescence polarization studies of the interaction of Escherichia coli protein synthesis initiation factor 3 with 30S ribosomal subunits

Biochemistry
J Weiel, J W Hershey

Abstract

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 subun...Continue Reading

References

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Citations

May 1, 1992·Biochimie·M G BubunenkoA T Gudkov
May 6, 2003·Protein Expression and Purification·Alexandra WolfrumNorbert Grillenbeck
Dec 13, 2007·Quarterly Reviews of Biophysics·Christopher S Fraser, Jennifer A Doudna
Aug 14, 2001·The EMBO Journal·D PetrelliC O Gualerzi
Apr 14, 1999·Proceedings of the National Academy of Sciences of the United States of America·J P McCutcheonJ Frank
May 24, 2003·Cold Spring Harbor Symposia on Quantitative Biology·A BashanA Yonath
Nov 10, 2005·Angewandte Chemie·Lucia GuerreroRudolf K Allemann
Feb 28, 2002·Methods : a Companion to Methods in Enzymology·M GluehmannA Yonath
Apr 8, 2006·Molecular Cell·Sean M Studer, Simpson Joseph
Apr 29, 2004·Chemistry & Biology·Elizebeth C TurnerRudolf K Allemann
Jun 26, 1990·Biochemistry·C O Gualerzi, C L Pon
Nov 3, 2005·Journal of the American Chemical Society·Lucia GuerreroRudolf K Allemann
Mar 1, 1995·Microbiological Reviews·E M Phizicky, S Fields

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