Abstract
A ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes has been identified using the following criteria: 1. A loss in the thiostrepton binding ability of the ribosome was correlated with the selective removal of ribosomal protein L11. This was achieved by a comparison of the thiostrepton binding ability of 50 S ribosomal subunits treated with 1 M NH4C1 and 50% ethanol at 37 degrees which still contained protein L11, and subunits treated successively at 0 degrees and 37 degrees in the same medium, from which protein L11 had been removed. 2. The thiostrepton binding ability of a ribosomal core containing only seven proteins, produced by treatment of 50 S subunits with 4 M LiC1, was fully restored by the rebinding of protein L11, obtained by Sephadex G-100 fractionation of the 1 M LiC1 split protein fraction from 50 S subunits. In addition, treatment of the 1 M LiC1 split protein fraction with an IgG specific for protein L11, uniquely inhibited the restoration of activity. 3. Thiostrepton binding to the 4 M LiC1 core, reconstituted with the 1 M LiC1 split protein fraction, was blocked by treatment with a monovalent antibody fragment (Fab) prepared against protein L11, but not by treatment with antibodies specific for the proteins of the 4 M LiC1 core. We conclude, therefore, that protein L11 is required for the ribosomal binding of thiostrepton.
Highlights
A ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes has been identified using the following criteria: 1. A loss in the thiostrepton binding ability of the ribosome was correlated with the selective removal of ribosomal protein Lll
Thiostrepton binding to the 4 M LiCl core, reconstituted with the 1 M LiCl split protein fraction, was blocked by treatment with a monovalent antibody fragment (Fab) prepared against protein Lll, but not by treatment with antibodies specific for the proteins of the 4 M LiCl core
The experiments described here were based on our previous observations’ that [YS]thiostrepton bound tightly, and stoichiometrically to Escherichia coli 50 S subunits, and were designed to identify the ribosomal protein responsible for thiostrepton binding
Summary
Preparation of Materials-The preparation of antisera against individual ribosomal proteins and the subsequent purification and characterization of IgGs and their corresponding Fab fragments from these sera were as described* [11,12,13,14]. Ribosomal core particles and split protein fractions were prepared by one of two methods. The ribosomal proteins from 50 S subunits and core particles were quantitatively extracted by the LiCl-urea method of Spitnik-Elson [16] These proteins and the split protein fractions were precipitated with 10% trichloroacetic acid, washed with ethanol and diethyl ether, resuspended in a small volume of 8 M urea, 10 mM NH,HCO,, and. To prepare RNA free of proteins, the RNA precipitates resulting from the extractions described above were washed three times with 3 M LiCl-4 M urea, resuspended in 8 M urea, and dialyzed until clear against a buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM MgCl,, and 50 mM. Ribosome Reconstitution-Reconstitution of LiCl core particles with split protein fractions was carried out at 0” by mixing both components together under the thiostrepton binding assay conditions listed above. The numbers used to identify the proteins on the gel electropherograms are according to the nomenclature of Kaltschmidt and Wittmann [19]
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