Amino acid starvation in an Escherichia coli auxotroph IV. The incorporation of [14C]leucine into cell fractions and its transfer between them

Abstract

Abstract A leucine-proline double auxotroph of Escherichia coli K-12 was starved of proline and allowed to incorporate [ 14 C]leucine. The labeled cells were reincubated in the absence of any amino acid, in the presence of non-radioactive leucine, or in the presence of proline. In each case the cells were harvested, lysed by sonic oscillation, and fractionated centrifugally and chemically. [ 14 C]Leucine was determined in a liquid scintillation spectrometer. 1. 1. [ 14 C]Leucine entered all cell fractions during proline starvation, even in 3 min at 0°, and about 100 times faster at 37°. 2. 2. On reincubation of the labeled cells with non-radioactive leucine during continued proline starvation, the bulk of the incorporated [ 14 C]leucine exchanged freely with the medium. Most of the counts associated with ribosomal protein were shifted out of that fraction, apparently into soluble protein. The latter behaved as an endproduct in that it never lost counts. 3. 3. On reincubation with proline in the absence of leucine about one-quarter of the counts in ribosomal protein were shifted into soluble protein, without any loss of counts from the acid-soluble pool. This is interpreted as representing the completion of those nascent protein molecules which required proline residues, and their removal from the templates. 4. 4. The findings are discussed in relation to the stepwise mechanisms of protein synthesis in E. coli .