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R Fast

Publications and source records attributed to R Fast.

7 recordsLinked to original sources
PubMed1987

Does streptomycin cause an error catastrophe?

We have examined the interpretation that streptomycin kills a bacterial culture by initiating the so-called error catastrophe. In particular, we asked whether the increased translational error rate induced by the antibiotic gives rise to an autocatalytic loss of functional fidelity of the devices responsible for gene expression, which ultimately causes the death of the culture. We have analyzed the performance characteristics of one of these devices, namely the ribosome in streptomycin-treated bacteria. We find that, although the treated ribosomes are constructed from error-containing proteins, they are not significantly different in elongation rate and fidelity from those ribosomes taken from untreated bacteria. We conclude that the bacteriocidal effect of streptomycin is not due to the initiation of an error catastrophe.

Dihydrostreptomycin Sulfate↗
PubMed1986

Association of RNA polymerase having increased Km for ATP and UTP with hyperexpression of the pyrB and pyrE genes of Salmonella typhimurium.

We investigated the transcription kinetics of RNA polymerase from an rpoBC mutant of Salmonella typhimurium which showed highly elevated, constitutive expression of the pyrB and pyrE genes as well as an increased cellular pool of UTP. When bacterial cultures containing an F' lac+ episome were induced for lac operon expression, the first active molecules of beta-galactosidase were formed with a delay of 73 +/- 3 s in rpo+ cells. The corresponding time was 104 to 125 s for cells carrying the rpoBC allele, indicating that this mutation causes a reduced RNA chain growth rate. In vitro the purified mutant RNA polymerase elongated transcripts of both T7 DNA and synthetic templates more slowly than the parental enzyme at a given concentration of nucleoside triphosphates. This defect was found to result from four- to sixfold-higher Km values for the saturation of the elongation site by ATP and UTP. The saturation kinetics of the RNA chain initiation step also seemed to be affected. The maximal elongation rate and Km for GTP and CTP were less influenced by the rpoBC mutation. Open complex formation at the promoters of T7 DNA and termination of the 7,100-nucleotide transcript showed no significant difference between the parental and mutant enzymes. Together with the phenotype of the rpoBC mutant, these results indicate that expression of pyrB and pyrE is regulated by the mRNA chain growth rate, which is controlled by the cellular UTP pool. The rate of gene expression is high when the saturation of RNA polymerase with UTP is low and vice versa.

Adenosine Triphosphate↗
PubMed1978

Isolation of Escherichia coli mutants with changed regulation of uracil uptake.

The incorporation of uracil into the pyrimidine ribonucleotide pools of Escherichia coli is strongly restricted under stringent conditions. Previously, we have suggested that this inhibition can be explained by the allosteric properties of uracil phosphoribosyltransferase. It has been proposed that this enzyme performs the uptake of uracil into the cell by transporting it across the cytoplasmic membrane, with the stimultaenous formation of UMP. To test this hypothesis it would be helpful to have mutants with changed regulation of uracil uptake, and in the present work, a method is introduced for the selection of such mutants. This method is based on phenotypic suppression of amber mutations by 5-fluorouracil (5FU). Mutants were isolated in an arginine-requiring strain of E. coli carrying an amber mutation in argI, the ornithine transcarbamylase gene. To facilitate the phenotypic rescue of this defective gene, mutants which overproduced ornithine transcarbamylase mRNA were isolated as a first step. The absence of exogenously added arginine causes stringent conditions, and phenotypic rescue by 5FU is, thus, prevented, unless the 5FU uptake mechanism is mutationally changed in such a manner that the drug is taken up into the cell. Three mutants in which the growth could be supported by 5FU in the absence of arginine were isolated. Two of them had acquired an increased ability to take up uracil under stringent conditions.

Arginine↗
PubMed1977

Biochemical mechanism of uracil uptake regulation in Escherichia coli B. Allosteric effects on uracil phosphoribosyltransferase under stringent conditions.

The regulation of uracil uptake in bacteria was studied in bacteriophage T4-infected cells, where host-specific, stable RNA synthesis is completely shut-off by phage, and where phage-specific RNA synthesis, which is not stringently regulated, could be followed by a continuous incorporation of uracil. This incorporation into phage RNA was found to be dependent on the allelic state of the rel gene and it was thus severely restricted under stringent conditions. This was not the case with adenine, which was incorported into RNA to almost the same extent under stringent and relaxed conditions, respectively. The inhibition of uracil uptake under proceeding RNA formation, which was furthermore found to be reversed by addition of chloramphenicol, indicated a specific mechanism governing the cellular entry of uracil. This is suggested to involve the allosteric regulation of uracil phosphoribosyltransferase (EC 2.4.2.9.). The enzyme was partially purified by ammonium sulfate precipitation and gel chromatography. The dependence on GDP and GTP as positive effectors was demonstrated. The stimulatory effect of GTP was abolished in vitro by the addition of guanosine 5'-diphosphate 3-diphosphate, which is known to accumulate during amino acid starvation in stringent bacteria. The reversible inactivation of the enzyme by dilution suggested a subunit structure of uracil phosphoribosyltransferase.

Adenine↗
PubMed1975

Regulation of early mRNA synthesis after bacteriophage T4 infection of Escherichia coli.

Regulation of T4-specific mRNA synthesis was studied during leucine starvation of a leucine-requiring stringent Escherichia coli B strain. This was done by imposing starvation prior to T4 infection and then letting RNA synthesis proceed for different time periods. Rifampin or streptolydigin was added to stop further RNA synthesis, and protein synthesis was restored by addition of leucine. Samples were withdrawn at different times, and the enzyme-forming capacities found that, during conditions which elicit the stringent response in uninfected bacteria, immediate early mRNA is not stringently regulated. This conclusion contradicts the earlier conclusion of others, obtained by measuring incorporation of radioactive uracil; this is explained by the observation of Edlin and Neuhard (1967), confirmed and extended by us to the T4-infected cell, that the incorporation of uracil into RNA of a stringent strain is virtually blocked by amino acid starvation, whereas that of adenine continues at 30 to 50% of the rate seen in the presence of the required amino acid.

Adenine↗