PMID: 3521729Jun 17, 1986

Binding of Escherichia coli protein synthesis initiation factor IF1 to 30S ribosomal subunits measured by fluorescence polarization

Biochemistry
F H Zucker, J W Hershey

Abstract

The interaction of initiation factor IF1 with 30S ribosomal subunits was measured quantitatively by fluorescence polarization. Purified IF1 was treated with 2-iminothiolane and N-[[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonic acid in order to prepare a covalent fluorescent derivative without eliminating positive charges on the protein required for biochemical activity. The fluorescent-labeled IF1 binds to 30S subunits and promotes the formation of N-formylmethionyl-tRNA complexes with 70S ribosomes. Analyses of mixtures of fluorescent-labeled IF1 and 30S ribosomal subunits with an SLM 4800 spectrofluorometer showed little change in fluorescence spectra or lifetimes upon binding, but a difference in polarization between free and bound forms is measurable. Bound to free ratios were calculated from polarization data and used in Scatchard plots to determine equilibrium binding constants and number of binding sites per ribosomal subunit. Competition between derivatized and nonderivatized forms of IF1 was quantified, and association constants for the native factor were determined: (5 +/- 1) X 10(5) M-1 with IF1 alone; (3.6 +/- 0.4) X 10(7) M-1 with IF3; (1.1 +/- 0.2) X 10(8) M-1 with IF2; (2.5 +/- 0.5) X 10(8) M-1 with both I...Continue Reading

References

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Citations

May 1, 1992·Biochimie·M G BubunenkoA T Gudkov
Aug 26, 2000·Progress in Biophysics and Molecular Biology·S Al-KaradaghiA Liljas
Dec 13, 2007·Quarterly Reviews of Biophysics·Christopher S Fraser, Jennifer A Doudna
May 9, 2012·Nature Structural & Molecular Biology·Pohl MilónMarina V Rodnina
Jan 20, 2004·European Journal of Biochemistry·Victor V CroitoruLeif A Isaksson
Jan 15, 1997·European Journal of Biochemistry·H P VornlocherM Sprinzl
Oct 14, 2014·Journal of Molecular Biology·Daniel D MacDougall, Ruben L Gonzalez
Sep 5, 2015·Nucleic Acids Research·Akanksha GoyalMarina V Rodnina
Jan 24, 2007·Molecular Microbiology·Gregory S Allen, Joachim Frank
Apr 8, 2006·Molecular Cell·Sean M Studer, Simpson Joseph
Mar 23, 2002·Cell·V Ramakrishnan
May 18, 1999·Journal of Molecular Biology·C SacerdotM Springer
Jun 26, 1990·Biochemistry·C O Gualerzi, C L Pon
Jun 22, 2000·Journal of Molecular Biology·K D Dahlquist, J D Puglisi
May 26, 2006·The EMBO Journal·Ayman AntounMåns Ehrenberg
May 24, 2003·Cold Spring Harbor Symposia on Quantitative Biology·C MayerU L RajBhandary

Related Concepts

Alkalescens-Dispar Group
Mathematics
Peptide Initiation Factors
Ribosomes
Fluorescence Spectroscopy
Eukaryotic Initiation Factor-2, gamma Subunit

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