Synthesis and breakdown of proteins in Escherichia coli during amino-acid starvation

Abstract

Using methionine- and proline-requiring strains of Escherichia coli ( rel + and rel − ), protein synthesis during amino-acid starvation was studied by analyzing the kinetics of radioactive labeling of proteins and the sedimentation and gel filtration distributions of the labeled products. The following results were obtained. 1. (1) The rate of protein synthesis drops within a few minutes after starvation to below 10% of the value observed before starvation (100% is the incorporation of 34.5 nmoles of amino acids/min/10 9 cells), and then continues to decrease slowly, such that it is reduced to 6% after 30 minutes. The average rate of peptide chain growth is reduced to the same extent from 17 to 1 amino acid per second per chain, suggesting that the movement of ribosomes along the messenger RNA molecules is intermittently arrested every time when ribosomes encounter codons requiring the incorporation of the missing amino acids. The average duration per arrest is estimated to be 12 seconds. 2. (2) During starvation, low molecular weight proteins are synthesized preferentially. The molecular weight distributions of proteins are consistent with the idea that during starvation incomplete peptide chains are prematurely released from the ribosomes with a probability of 4% per arrest and that the probability for a peptide to be completed decreases exponentially with its molecular weight. 3. (3) Of the proteins synthesized during starvation 35% are unstable (half lifetime = 3 min). The average molecular weight of the unstable proteins is below 20,000 daltons, suggesting that the instability reflects an increased susceptibility of the incomplete peptide fragments to proteolytic attack. 4. (4) The synthesis of active β-galactosidase is reduced to 0.7% during starvation and the enzyme is stable. This more-than-average reduction of enzyme synthesis is not due to a reduced synthesis of β-galactosidase messenger RNA, but is consistent with the idea of the synthesis of peptide fragments. 5. (5) Essentially the same results were obtained with rel + and rel − bacteria, suggesting that the rel + gene product does not interfere with the movement of ribosomes along the messenger.