Hazel D. Barner

Protein synthesis and RNA turnover in a pyrimidine-deficient bacterium.

Abstract An auxotrophic mutant of E. coli strain 15 has been isolated possessing thymine and uracil requirements. Lack of oxogenous thymine prevents DNA synthesis; lack of oxogenous uracil inhibits net RNA synthesis. Lack of both pyrimidines prevents net nucleic acid synthesis, but does not prevent protein synthesis. The protein synthesized under such conditions may be directed by induction and be a specific enzyme such as xylose isomerase. Despite the absence of net nucleic acid synthesis in pyrimidine deficiency, a fraction of the RNA was found to have a marked turnover in which preformed uracil of RNA was released and reincorporated with newly formed ribose generated in the presence of glucose-14C. The very low isotope content of thymidine under comparable conditions indicates a negligible turnover of DNA. The possible significance of the RNA turnover has been discussed.

Some physiological and genetic properties of a strain of Escherichia coli requiring thymine, arginine and uracil

A polyauxotrophic mutant of E. coli strain 15 has been studied. The organism requires thymine, uracil and arganine for normal growth and multiplication. The formation of citrulline and ureidosuccinate is blocked almost completely. Nevertheless, significant although depressed amounts of both ornithine and aspartate transcarbamylases are readily detectable in cell-free extracts of the organism. The bacterium can be induced to synthesize xybose isomerase in the presence of exogenous arginine and in the absence of exogenous uracil, as in strain 15T−U−. Relatively extensive peptide synthesis is obtained under these conditions. Analysis of the observed RNA metabolism suggests a turnover of part of the RNA and a greater conservation of the bases than of the ribose. Most of the turnover appears to depend on an exogenous supply of arginine, as does the function of enzyme formation. Reversion to arginine and uracil independence occurs in a single genetic step. Thymine starvation increase the percentage of revertants in the culture before the bacteria die as a result of unbalanced growth.

The lethality of streptomycin and the stimulation of RNA synthesis in the absence of protein synthesis.

The action of streptomycin and related drugs has been studied with thymineless, arginineless and uracilless strains of Escherichia coli. Many experiments were performed in the absence of arginine, a condition shown to reduce 35SO42−incorporation (mainly protein synthesis) to 0·5% and less of the rate of incorporation during growth. In the absence of arginine and in the presence of thymine, under conditions in which the rate of RNA synthesis is 10 to 15% of that during normal growth, streptomycin initially inhibits and then markedly stimulates RNA synthesis. In a wide variety of conditions, the inception of stimulation coincides closely with the inception of lethality. Also the rate of stimulated synthesis of RNA correlates well with the rate of loss of capacity to form colonies. Such a stimulation of RNA synthesis and correlation with lethality also occurs in the presence of neomycin and kanamycin. The stimulation does not occur in a derived streptomycin-resistant strain in which streptomycin does inhibit slightly the basal level of RNA synthesis occurring in the absence of protein synthesis. Anaerobiosis, which protects against the lethal action of streptomycin, also prevents the stimulation of RNA synthesis, although the internal concentrations of phosphates and of streptomycin during anaerobiosis were not significantly changed as compared to the concentrations of these substances during aerobiosis. Reduction of the phosphate concentration in a medium containing streptomycin reduced the lag before both the beginning of killing and the stimulation of RNA synthesis, and increased the degree of the latter function. Indeed under these conditions, the stimulation produced by streptomycin approached the stimulated rate of synthesis of RNA produced on addition of chloramphenicol. The RNA made under the influence of streptomycin was partially associated with the ribosomal fraction and had a rapid but incomplete turnover; however, the partial degradation of this RNA in the absence of the antibiotic did not permit the restoration of apparently killed bacteria to viability. The incorporation of fluorouracil in the presence or absence of uracil was not lethal in the absence of streptomycin. In the presence of the antibiotic, lethality was increased by the analog, concomitant with the stimulated incorporation of the analog into the new type of RNA. At the beginning of stimulation of RNA synthesis, the capacity to incorporate arginine is reduced about 50%. The formation of the RNA stimulated by the antibiotic in the absence of protein synthesis (measured by uptake of 35SO42−) is coincident with a rapid reduction of the capacity to incorporate arginine to less than 5% of the normal rate. Thus physiological lethality, i.e. the irreversible loss of the ability to make proteins in the absence of streptomycin, occurs without protein synthesis.