Observations on Molecular Weight Determinations on Polyacrylamide Gel

3 – Proteins and Sodium Dodecyl Sulfate: Molecular Weight Determination on Polyacrylamide Gels and Related Procedures

An assembly map of 30S ribosomal subunits has been constructed. The assembly reaction is sequential and cooperative. The map may reflect the topological relationships among ribosomal proteins in the ribosomal structure.

Assembly Mapping of 30S Ribosomal Proteins from E. coli

The streptomycin locus of E. coli specifies a 30S ribosomal protein which determines the sensitivity of the 30S subunit to streptomycin and streptomycin induced errors of translation.

Identification and Functional Characterization of the Protein controlled by the Streptomycin-resistant Locus in E. coli

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.

https://www.pnas.org/content/pnas/67/3/1276.full.pdf

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

Ribosomal Proteins of E. coli: Stoichiometry and Implications for Ribosome Structure

Ribosomal protein from E. coli separates during gel electrophoresis into a large number of components.'-3 A variety of experiments have been done to determine if the apparent complexity might be misleading and be caused by interactions among a smaller number of components. No evidence has been found in favor of this hypothesis.\"' 3-7 The most likely conclusion is that the multiple components seen on gel electrophoresis correspond to different protein molecules in the ribosome population. The possibility remains, however, that the different electrophoretic components are closely related chemically and are only slight modifications or combinations of one or a few primary structures. This view has been favored by success in elucidating the general mechanism of protein synthesis while considering the ribosome a passive structure or template, and by the plausible analogy between ribosome and virus structure. On the other hand, the recent demonstration that specific fractions of ribosomal protein restore in vitro activity to protein depleted particles6-9 and the evidence indicating that peptidyl transferase is an integral part of the 50S ribosome'0'-2 are more consistent with the concept of the ribosome as an enzyme complex composed of functionally specific, chemically distinct proteins. The experiments presented here were designed to distinguish between these alternatives using two approaches. (1) The number of tryptic peptides of the total protein containing sulfur was examined. The relationship of this number to the total calculated number of sulfur-containing residues should reflect the extent to which common sequences occur. (2) Several pure proteins have been isolated and tryptic fingerprints and amino acid compositions compared. Evidence of both types leads to the conclusion that the ribosomal proteins are chemically heterogeneous. Materials and Methods.-Early and late log phase E. coli B cells were purchased from General Biochemicals, Chagrin Falls, Ohio. Cell extracts were prepared by homogenization with glass beads\"3 or with a Hughes pressure cell. Ribosomes and ribosomal subunits were prepared by standard methods.\"' 15 Ribosomal proteins were extracted with 66 per cent acetic acid.'6 Protein concentrations were determined by the Folin method.'7 N-terminal groups were found by the dinitrofluorobenzene (DNFB) method.'8 19 Acrylamide gel electrophoresis and radioautography were done as described by Traut.3 S36 MRE-60021 cells were grown as described previously.3 Cysteine was labeled selectively by including 10 ,g/ml of nonradioactive methionine in the medium. The exclusion of S35 from methionine was confirmed by analysis of acid hydrolysates by paper electrophoresis at pH 1.9 followed by radioautography. Carboxymethyl cellulose was obtained from Calbiochem (Los Angeles) and from W. and R. Balston, Ltd. (England). Performic acid oxidation was carried out according to Nathans1° and carboxymethylation according to Spackman et al.26 Purification of the proteins was achieved on carboxy-

A. Tissières

Papers

Ribosomal Proteins of E. coli: Stoichiometry and Implications for Ribosome Structure

Ribosomal proteins of Escherichia coli. I. Demonstration of different primary structures