Mutants defective in utilization of uracil at low concentrations have been isolated and characterized. The mutations in question (uraA) map close to the upp gene encoding uracil phosphoribosyltransferase. By complementation analysis, a plasmid that complements the uraA mutation has been isolated. The uraA gene was shown to be the second gene in a bicistronic operon with upp as the promoter proximal gene. The nucleotide sequence of the gene was determined, and the gene encodes a hydrophobic membrane protein with a calculated M r of 45,030. The UraA protein has been identified in sodium dodecyl sulfate-polyacrylamide gels in the membrane fraction of minicells harboring the uraA plasmids.

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 13-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.

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Exogenous uracil was shown to be taken up efficiently into ribonucleotide pools a t all times during the cycle of T4 phage infection, in which stable host RNA synthesis is known to be turned off. This uptake was shown to be effected by ribonucleotide pool expansion and a net increase in mRNA content after phage infection. By comparing different phage mutants, the draining of ribonucleotide pools into DNA synthesis by ribotide reduction, was also found to be instrumental for pool labeling from exogenous uracil. The UDPG pool expanded as much as 3-fold during DNA negative mutant infection, presumably as a result of absent DNA glucosylation.From measurements of specific activities of nucleotides by double labeling and thin-layer chromatography, it was concluded that the incorporation into RNA of a radioactive, exogenous base can be reliably used for following T4 phage transcription.It was stated by Nierlich [1,2], that in bacteria a large synthesis of labile RNA could go undetected or be greatly underestimated by RNA base incorporation experiments under conditions of no synthesis of stable RNA. This statement was based on the demonstration that the entry of an exogenous radioactive base into the intracellular nucleotide pools was not facilitated by exchange reactions between nucleotides and free base or by nucleotide pool expansion, and that the labeling of the pools from the exogenous base was relatively slow [3]. The conclusion was that the entry of isotope is not faster than the removal of pool components for net RNA synthesis.In the case of stringent regulation of RNA synthesis in bacteria starved for amino acids, a ceasing synthesis of stable RNA could thus make synthesis of labile mRNA undetectable by incorporation experiments [a].