Studies on the structure and expression of Escherichia coli pyrC, pyrD and pyrF using the cloned genes

The upp gene coding for uracil phosphoribosyltransferase was subcloned on a 5‐kb EcoRI restriction fragment along with the purMN operon. By a combination of complementation, deletion and minicell analyses, the upp gene was located adjacent to and divergently transcribed from the purMN operon. All three gene products could be identified in minicell extracts. The cloned upp gene shows an elevated expression upon uracil starvation. The nucleotide sequence and transcription start of the gene were determined. The sequence yields an open reading frame of 624 nucleotides encoding a protein of 22.5 kDa which is in agreement with the previously determined subunit Mr of the purified enzyme. A putative 5‐phosphoribosyl‐α‐1‐diphosphate (PRPP) binding site has been identified which is similar to the PRPP binding site of the yeast uracil phosphoribosyltransferase.

Section: Gene Expression In Minicellsmentioning

confidence: 99%

Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12

Andersen¹,

Smith²,

Mygind³

1992

“…Minicells, obtained from cultures of BD1854 containing the desired plasmid, were labeled with [35S]methionine and extracted [21]. The labeled extracts were analyzed by electrophoresis through 12.5% polyacrylamide/SDS gels.…”

Section: Plasmid-coded Proteinsmentioning

confidence: 99%

Nucleotide sequence of the carA gene and regulation of the carAB operon in Salmonella typhimurium

Kilstrup¹,

Lu²,

Abdelal³

et al. 1988

The carAB operon of Salmonella typhimurium encoding carbamoyl-phosphate synthetase (CPSase) has been cloned, and the nucleotide sequence of the first gene of the operon, carA, together with 760 base pairs of the 5'-flanking region was determined. The product of the carA gene is the small subunit of CPSase. It catalyzes the transfer of the amide group from glutamine to an NH,-site on the heavy subunit. Primer extension and S1 nuclease mapping of in vivo carAB transcripts revealed that transcription is similar to that of Escherichia co/i [Piette, J. et al. (1984) Proc. Nut1 Acad. Sci. U S A 81, 4134-41381 in its initiation at two promoters, PI and P2, controlled by pyrimidines and arginine, respectively. The arginine control is mediated through binding to the arginine repressor (argR). The involvement of titratable regulatory elements is indicated by the escape from both arginine and pyrimidine control, when the operon is present in multicopies on a plasmid. Measurements of CPSase levels in mutants which allows independent manipulation of the intracellular uracil and cytosine nucleotide pools show, that both uracil and cytosine nucleotides are required for full repression and that limitation of either nucleotide results in derepression of CPSase synthesis. Deletion analyses indicate that regions upstream of the PI promoter are required for normal expression from this promoter but not from P2.Carbamoyl-phosphate synthetase (CPSase) encoded by the carAB (pyrA) operon in Salmonella typhimurium [3] and Escherichia coli [4], catalyzes the synthesis of carbamoyl-phosphate from ATP, bicarbonate and glutamine. It plays a dual role in metabolism by supplying carbamoyl phosphate for both the pyrimidine and the arginine biosynthetic pathways. In accordance with its physiological function, CPSase is allosterically regulated by intermediates of both pathways. The pyrimidine nucleotide UMP inhibits CPSase activity while ornithine, an intermediate in the arginine biosynthetic pathway stimulates it [5].The enzyme from S. typhimurium consists of two nonidentical subunits. The light subunit (Mr = 45000), encoded by the first gene in the operon (carA), converts the amide nitrogen of glutamine to ammonia. The heavy subunit ( M , = IlOOOO), encoded by the second gene of the operon (carB), catalyzes the formation of carbamoyl-phosphate from ammonia, carbon dioxide and ATP, it also contains the binding sites for ornithine. The ammonia-dependent activity of the isolated heavy subunit is only slightly inhibited by high concentrations of UMP and the reconstituted holoenzyme regains sensitivity to this feedback inhibitor [3]. [I]. ,In S. typhimuriurn the homologous locus is designated pyrA [2]. In order to be consistent we propose that the E. coli nomenclature is adopted for the genes encoding S. typhimurium carbamoyl-phosphate synthetase.Expression of carAB is subject to cumulative repression by pyrimidines and arginine. In E. coli [4, 6, 71 transcription of the operon was shown to be initiated at tandem promoters. Transcription from th...

“…Protein synthesis in minicells was performed with [35S]methionine and analysed in 12.5% polyacrylamide gels, as described previously [24]. Low-molecular-mass markers from Bio-Rad were included on each gel.…”

Section: Protein Synthesis In Mniicellsmentioning

confidence: 99%

Attenuation in the rph‐pyrE operon of Escherichia coli and processing of the dicistronic mRNA

Andersen

Poulsen

Jensen

1992

Self Cite

We have substituted on a plasmid the native promoter of the Escherichiu coli rph-pyrE operon with an inducible transcription-initiation signal. The plasmid was used to study the mRNA chains derived from the operon at different intracellular concentrations of UTP and as a function of time following induction of transcription. The results showed that dicistronic rph-pyrE mRNA was formed when the UTP pool was low, and that a monocistronic rph mRNA was the major transcription product in high-UTP pools, thus supporting an UTP-controlled attenuation mechanism for regulation of pyrE gene expression. However, the dicistronic rph-pyrE transcript was rapidly processed into two monocistronic mRNA units, and a cleavage site was mapped near the attenuator in the intercistronic region, close to the site where transcription was terminated in high-UTP pools. Furthermore, the major 3' end of the pyrE mRNA was mapped near a palindromic structure of similarity to the family of repetitive extragenic palindromic sequences, 35 nucleotide residues after the stop codon of thepyrE gene.The pyrE gene encodes the pyrimidine-biosynthetic enzyme orotate phosphoribosyltransferase, which is responsible for formation of the glycosidic bond of pyrimidine nucleotides. ThepyrE gene maps at 81.7 min on the chromosome of Escherichia coli and is co-transcribed with a gene, provisionally called orfE, since the function of the 238-amino-acid protein product was unknown [l, 21. However, it was recently discovered that the OrfE protein is identical to the polynucleotide: orthophosphate nucleotidyltrdnsferase (RNase PH) [3], which is involved in the trimming of the 3' ends of tRNA precursors [4] and, for that reason, the ocfE gene has been renamed rph [3].Previous studies have revealed that the level of orotate phosphoribosyltransferase is inversely correlated with the size of the intracellular pools of UTP [5] and GTP 16, 71. The mechanistic basis for this regulation is a UTP/GTP-controlled attenuation of transcription in the intercistronic rph-pyrE region, taking place in such a way that the frequency of transcriptional read-through, past the attenuator and into the pyrE gene, increases when the UTP pool (or the GTP pool) decreases, and vice versa [2, 81. In addition, it is a prerequisite for attenuator regulation that ribosomes translating the rph cistron are allowed to follow RNA polymerase so closely that [16] all influence the attenuation frequency, it appears to be the degree of coupling between the transcribing RNA polymerase and the leading ribosome as a function of nucleoside triphosphates which determines the level ofpyrE gene expression [17].In previous studies, this attenuation system was investigated by using either lucZ or gulK fusions to different parts of the rph-pyrE operon. To obtain more direct evidence for the proposed regulation mechanism, we decided to study the rph-pyrE mRNA species directly by Northern hybridization and to map the end points of the attenuated and non-attenuated transcripts. For that purpose a plasmid was constru...