Summary
A mutant strain of Eschrichia coli that is temperature-sensitive for growth stopped protein biosynthesis at 43° C after a brief lag (Fig. 1). Cell-free extracts from the strain showed no specific defect in aminoacyl-tRNA synthetases, binding initiator tRNA to ribosomes (Table 1), protein chain elongation (Tables 2, 5) or protein chain termination (Tables 3, 4) at high temperature.
The partially purified enzyme peptidyl-tRNA hydrolase, however, was temperature-sensitive (Table 6); the mutant hydrolase was inactivated rapidly at 43° C (Table 7). Mixing experiments ruled out the presence, in the mutant enzyme preparation, of an inhibitor and also demonstrated, on the mutant enzyme, a protective effect by wild type enzyme that was not shown by general coli proteins (Tables 8, 9).
Interrupted mating allowed the temperature-sensitive growth phenotype to be mapped near to and before trp (Figs. 4, 5). Co-transsduction, mediated by bacteriophage P1, with trp + (frequency 7.5%) located the marker at 24 min on the coli map. All transductants for temperature-sensitive growth also had temperature-sensitive peptidyl-tRNA hydrolase activity in crude sonicates (Table 10). We provisionally conclude that the temperature-sensitive protein synthesis and growth are caused by a single genetic change in the structural gene (pth) for peptidyl-tRNA hydrolase.
After shift to 43° C the polysomes of the mutant cells broke down into 70S particles (Figs. 2, 3). A defect in protein biosynthesis thus appeared to be located after termination and before reformation of new polysomes.
The metabolic role of peptidyl-tRNA hydrolase is discussed in the light of these experiments.
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Communicated by M. Nomura
Journal paper No. J-7465 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 1747.
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Menninger, J.R., Walker, C., Tan, P.F. et al. Studies on the metabolic role of peptidyl-tRNA hydrolase. Molec. Gen. Genet. 121, 307–324 (1973). https://doi.org/10.1007/BF00433230
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DOI: https://doi.org/10.1007/BF00433230