Further evidence for the involvement of charged tRNA and guanosine tetraphosphate in the control of protein degradation in Escherichia coli

St John, A.C.; Conklin, K.; Rosenthal, E.; Goldberg, A.L.

Journal of Biological Chemistry 253(11): 3945-3951


ISSN/ISBN: 0021-9258
PMID: 348699
Accession: 068526755

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Previous studies suggested that protein degradation, like RNA synthesis, is regulated in rel A+ strains by the level of charged tRNA, which determines the rate of synthesis of guanosine tetraphosphate (ppGpp). Because these ideas were questioned recently, protein degradation was re-examined in isogenic rel A+ and rel A- E. coli strains (NF536/NF537) containing a temperature-sensitive valyl-tRNA synthetase. At 30.degree. C, growth occurred and rates of protein breakdown were similar in the 2 strains. At 36.degree. C or higher temperatures, the valyl-tRNA synthetase was inactivated, ppGpp accumulated and net RNA synthesis was markedly inhibited in the rel A+, but not in the rel A- cells. In addition, at 36.degree. C and 39.degree. C, protein degradation was several times faster in the rel A+ than in the rel A- strain. Similar effects were obtained with another rel A+/rel A- pair carrying a distinct temperature-sensitive mutation. Thus, the absence of valyl-tRNA which results in the accumulation of ppGpp in rel A+ strains stimulated protein degradation. At 42.degree. C, overall rates of protein breakdown were rapid in both rel A+ and rel A- cells and thus were independent of the levels of uncharged tRNA or ppGpp. These nonspecific effects of high temperatures can account for the discrepant conclusions by others. Further evidence that the lack of a single charged tRNA species stimulates protein breakdown was obtained in experiments using E. coli K12 treated with serine hydroaxamate, a compettitive inhibitor of seryl-tRNA synthetase. Protein synthesis is not required for the stimulation of protein degradation upon starvation of rel A+ cells for valyl-tRNA. Although chloramphenicol decreases proteolysis in wild type cells during amino acid starvation, this effect probably results from the concomitant accumulation of charged tRNA and the consequent suppression of ppGpp synthesis. Thus, addition of chloramphenicol to strain NF536 at 39.degree. C, where the inactivation of the valyl-tRNA synthetase prevented accumulation of charged tRNA, did not decrease ppGpp or protein catabolism to basal levels. Tetracycline, which directly prevents ppGpp synthesis, reduced ppGpp and protein catabolism in rel A+ cells to the levels found in rel A- cells. Thus, the stimulation of proteolysis seems to require the continued production of ppGpp. Additional experiments examined the possible involvement in the control of protein catabolism of other pleiotropic regulatory systems known to play important roles under nutrient-poor conditions. The rate of protein degradation in growing cells is not influenced by and the enhancement of this process during starvation does not require cyclic AMP or the active form of glutamine synthetase.