Synthesis and Translation of Ribopolymers in Escherichia coli Made Permeable by Treatment in High Sucrose Concentration

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Abstract
An Escherichia coli“permeabilized” cell system obtained by treatment in high sucrose concentration, because of its depletion from endogenous metabolites, its low nuclease, phosphatase and phosphokinase content, catalyzes a very efficient polymerisation of the common ribonucleoside diphosphates (UDP, CDP, ADP and GDP) presumably through the polynucleotide phosphorylase retained within the cells after osmotic shock. Accordingly incorporation of nucleotides into polymers is insensitive to actinomycin D and does not take place in the presence of Mg2+ions with E. coli Q13 a strain in which the polynucleotide phosphorylase is a Mn‐dependent enzyme. Polymers containing U, C, G, or A as the only constituting nucleosides have been found to accumulate for several hours at a linear rate, in the supernatant phase from osmotically shocked E. coli D10. If, following the arrest of polymer formation the amino acid coded for by the corresponding triplet is added to the incubation medium together with an energy‐yielding system, a very efficient incorporation of this amino acid (and of this amino acid only) into an acid‐precipitable material takes place. The resulting product (presumably an homopolypeptide) is associated with the cell bodies. Translation of the preformed polyribonucleotide is concomitant with its nucleolytic cleavage by a K‐dependent nuclease.