Ribosomal Proteins, XII. Number of Proteins in Small and Large Ribosomal Subunits of Escherichia coli as Determined by Two-Dimensional Gel Electrophoresis

Abstract

Abstract. Two-dimensional gel electrophoresis separates all of the component proteins of the ribosomal subunits of Escherichia coli. This method shows 21 proteins in the 30S, and 34 proteins in the 50S, subunit.Waller' showed that the ribosomes of Escherichia coli contain a large number of protein components. Determination of the exact number of ribosomal proteins is important for precise knowledge of the structure and function of the ribosomes. Combinations of two or more conventional methods, such as disk electrophoresis on acrylamide gels under different conditions, ion exchange, and molecular sieve chromatography, indicate that the 30S and 50S ribosomal subunits contain 20 and 30-35 proteins, respectively.2-6 We have recently developed a two-dimensional (2D) polyacrylamide gel electrophoresis procedure' by means of which all of the proteins of the ribosomal subunits can be rapidly and reproducibly separated in one operation. This paper describes the results of this procedure for the separation of proteins derived from 30S and 50S subunits, as well as 70S ribosomes, of E. coli. We find that the 30S subunits contain 21 proteins, and the 50S subunit as many as 34 proteins. A numbering system is proposed based on the position of the protein spots on the 2D electropherogram.Materials and Methods. Electrophoresis: Two-dimensional electrophoresis was performed as described.7 The acrylamide concentration in the first dimension was 8%, except in a few cases where it was 4%. Tris-EDTA-boric acid buffer, pH 8.6, and less frequently, pH 9.6, was used. The electrophoretic conditions in the second dimension were always 18% acrylamide gel in acetate buffer, pH 4.6.Preparation of the proteins: The 70S ribosomes obtained by differential centrifugation were washed with 0.5 M NH4Cl, 1 mM Mg++. Subunits were separated by sucrose gradient centrifugation in a zonal rotor, B XV. Proteins were extracted from ribosomes by treatment with 67% acetic acid in the presence of 30 mM Mg++ (ref. 4). The RNA precipitate was washed with extracting solution for more quantitative extraction of the proteins. No further proteins were detected in the RNA precipitate after washing with 67% acetic acid in the presence of higher salt concentrations. The proteins were lyophilized and dissolved in the components of the sample gel. The solubility of the proteins improved when the extracted proteins, instead of being lyophilized, were directly dialyzed against the gel components. When mentioned in the text, the proteins were oxidized with performic acid or reduced and alkylated with iodoacetamide.8Results and Discussion. The protein mixtures extracted from a large number of ribosome preparations were compared by two-dimensional electrophoresis.

Comparative studies on ribosomal proteins of Escherichia coli 113‐3 and strains B, C and K12

“…Under the electrophoretic conditions used, proteins L 4 and L 10 show an electroneutral character (figs.3 and 4). Protein L 21, which according to Kaltschmidt and Wittmann [3] is slightly acidic, in the material from Kt2 and B strains in our electrophoretic conditions was electroneutral, while in that from the 113-3 strain was slightly basic (fig.4).…”

“…The maps of the ribosomal proteins from wild strains were made for identification and comparison, as our conditions of 2D electrophoresis are somewhat different from those used by Kaltschmidt and Wittmann [3]. The map of ribosomal proteins published by those authors and their numbering system were the form adopted by us in classifying the proteins from E. coli 113-3 ribosomes.…”

“…Four of them have their isoelectric points at pH < 8. separate as two clearly discernible spots both in the material derived from wild strains of bacteria as well from the mutant strain. According to Kaltschmidt et al [3], these proteins become separated in the course of the electrophoresis at pH 9.6. Under the electrophoretic conditions used, proteins L 4 and L 10 show an electroneutral character (figs.3 and 4).…”

“…coli ribosomes [4]. Kaltschmidt and Wittmann [3] described the results of this procedure for the separation of proteins derived from 30 S and 50 S subunits, as well as 70 S ribosomes ofE.. coli. They found that the 30 S subunits contain 21 proteins, at the 50 S subunit as many as 34 proteins.…”

“…The development of two-dimensional polyacrylamide gel electrophoresis procedure [2,3] of separation of the extraordinarily complex mixture of ribosomal proteins has stimulated detailed studies on the structure and function ofE. coli ribosomes [4].…”