Proteins on ribosome surface: measurements of protein exposure by hot tritium bombardment technique

Abstract

The hot tritium bombardment technique [Goldanskii, V. I., Kashirin, I. A., Shishkov, A. V., Baratova, L. A. & Grebenshchikov, N. I. (1988) J. Mol. Biol. 201,567-574] has been applied to measure the exposure of proteins on the ribosomal surface. The technique is based on replacement of hydrogen by high energy tritium atoms in thin surface layer of macromolecules. Quantitation of tritium radioactivity of each protein has revealed that proteins S1, S4, S5, S7, S18, S20, and S21 of the small subunit, and proteins L7/L12, L9, L10, L11, L16, L17, L24, and L27 of the large subunit are well exposed on the surface of the Escherichia coli 70 S ribosome. Proteins S8, S10, S12, S16, S17, L14, L20, L29, L30, L31, L32, L33, and L34 have virtually no groups exposed on the ribosomal surface. The remaining proteins are found to be exposed to lesser degree than the well exposed ones. No additional ribosomal proteins was exposed upon dissociation of ribosomes into subunits, thus indicating the absence of proteins on intersubunit contacting surfaces.

Figure 1

Labeling of total ribosomal protein by hot tritium bombardment. Proteins were denatured with 67% (▿) or with 5% (•) acetic acid. Tritium radioactivity of each individual protein is plotted against its molecular mass.

Figure 2

Separation of individual ribosomal proteins by two-dimensional gel electrophoresis: photograph of Coomassie-stained gel (A) and fluorogram of the same gel (B). Proteins were extracted from 70S ribosomes labeled at 10 mM Mg²⁺ (buffer C). The gel contains 150 μg of total ribosomal protein.

Figure 3

Labeling of nondissociated 70S ribosomes at 10 mM Mg²⁺ (buffer C). Tritium radioactivity of each protein is shown by three bars representing three independent experiments. Black bars, the well-exposed proteins; grey bars, the poorly exposed proteins; dark gray bars, the moderately exposed proteins. (A) Proteins of the 30S subunit; (B) proteins of the 50S subunit.

Figure 4

Labeling of the equimolar mixture of ribosomal subunits at 1 mM Mg²⁺ (buffer D). Radioactivity of each protein is shown by bars in percents of the total radioactivity found in all the protein spots on the gel. Gray bars, proteins labeled within subunits of dissociated ribosomes; black bars, proteins labeled within the nondissociated ribosomes at 10 mM Mg²⁺. Each bar represents an independent experiment. (A) Proteins of the 30S subunit, and (B) proteins of the 50S subunit.