SYNTHESIS OF NASCENT PROTEIN BY RIBOSOMES IN ESCHERICHIA COLI

Abstract

Introduction.-Cells and tissues of all kinds of living organisms have been found to contain ribonucleic acid (RNA) and protein both as separate constituents and as complexes in the form of ribonucleoprotein particles. From a variety of experimnental data it has been inferred that these particles are probably intimately involved in the processes of protein synthesis. The term microsome was originally applied to the portion of disrupted animal tissue which sedimented between 20,000 and 100,000 X g. It was found to contain most of theRNA. Later it was established that microsome preparations consisted of an association of ribonucleoprotein particles with fragments of the endoplasmic reticulum and techniques were evolved for removing the latter to yield particles which contained little else besides RNA and protein (see review by Palade). Similar particles have also been isolated from bacteria and the term ribosome has been suggested for the purified particles which are essentially free from lipid and other extraneous matter. In bacteria, depending on the cultural conditions, up to 80 per cent of the RNA may exist in varying sized ribonucleoprotein particles. These range in sedimentation constants from about 20S to 100S and some preparations have about 2 amino acid residues per ribonucleotide. The distribution among the various classes found in Escherichia coli depends on the cultural conditions and the conditions of cel breakage. Exponentially growing organisms possess ribosomes with sedimentation constants approximately 15-20S, 30S, 50S, 70S, 85S, 100S. The last is usually a minor component or absent except in resting cells and the 70S and/or 85S particles predominate over the smaller sizes. The sedimentation constant of the particle which we refer to as 85S does in fact vary between 70S and 100S depending on the magnesium concentrations. Separation of the various particles into homogeneous fractions presents considerable difficulties if attempted by simple differential centrifugation. We have found, however, that centrifugation of cell juices layered on sucrose solutions in the swinging bucket rotor of the Spinco can give a useful resolution of all classes of ribosomes seen in the analytical ultracentrifuge. In addition the isolated fractions are available for analysis. This technique has been used to observe the incorporation of radioactive tracers into the ribosomes and the soluble protein of E.coli.