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Binding of Escherichia coli protein synthesis initiation factor IF1 to 30S ribosomal subunits measured by fluorescence polarization. 1986

F H Zucker, and J W Hershey

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 IF2 and IF3. In all cases, 0.9-1.1 binding sites per 30S subunit were detected. Divalent cations have little effect on affinities, whereas increasing monovalent cations inhibit binding. On the basis of the association constants, we predict that greater than 90% of native 30S subunits are complexed with all three initiation factors in intact bacterial cells.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008433 Mathematics The deductive study of shape, quantity, and dependence. (From McGraw-Hill Dictionary of Scientific and Technical Terms, 6th ed) Mathematic
D008954 Models, Biological Theoretical representations that simulate the behavior or activity of biological processes or diseases. For disease models in living animals, DISEASE MODELS, ANIMAL is available. Biological models include the use of mathematical equations, computers, and other electronic equipment. Biological Model,Biological Models,Model, Biological,Models, Biologic,Biologic Model,Biologic Models,Model, Biologic
D010448 Peptide Initiation Factors Protein factors uniquely required during the initiation phase of protein synthesis in GENETIC TRANSLATION. Initiation Factors,Initiation Factor,Factors, Peptide Initiation,Initiation Factors, Peptide
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D012270 Ribosomes Multicomponent ribonucleoprotein structures found in the CYTOPLASM of all cells, and in MITOCHONDRIA, and PLASTIDS. They function in PROTEIN BIOSYNTHESIS via GENETIC TRANSLATION. Ribosome
D013050 Spectrometry, Fluorescence Measurement of the intensity and quality of fluorescence. Fluorescence Spectrophotometry,Fluorescence Spectroscopy,Spectrofluorometry,Fluorescence Spectrometry,Spectrophotometry, Fluorescence,Spectroscopy, Fluorescence
D015852 Eukaryotic Initiation Factor-2 Eukaryotic initiation factor of protein synthesis. In higher eukaryotes the factor consists of three subunits: alpha, beta, and gamma. As initiation proceeds, eIF-2 forms a ternary complex with Met-tRNAi and GTP. EIF-2,Peptide Initiation Factor EIF-2,EIF-2 alpha,EIF-2 beta,EIF-2 gamma,EIF-2alpha,EIF-2beta,EIF-2gamma,EIF2,Eukaryotic Initiation Factor-2, alpha Subunit,Eukaryotic Initiation Factor-2, beta Subunit,Eukaryotic Initiation Factor-2, gamma Subunit,Eukaryotic Peptide Initiation Factor-2,EIF 2,EIF 2 alpha,EIF 2 beta,EIF 2 gamma,EIF 2alpha,EIF 2beta,EIF 2gamma,Eukaryotic Initiation Factor 2,Eukaryotic Initiation Factor 2, alpha Subunit,Eukaryotic Initiation Factor 2, beta Subunit,Eukaryotic Initiation Factor 2, gamma Subunit,Eukaryotic Peptide Initiation Factor 2,Initiation Factor-2, Eukaryotic,Peptide Initiation Factor EIF 2

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