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

Abstract

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...

Allosteric properties of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermoacidophilic archaeon Sulfolobus solfataricus

“…Guanidinium hydrochloride and other fine chemicals were from Sigma, Serva (Heidelberg, Germany), Merck (Darmstadt, Germany), Calbiochem (EMD Biosciences Inc., Darmstadt, Germany) or Baker (Deventer, the Netherlands). The bacterial strain NF1830, E.coli K‐12 ( recA1/F'lacI q1 lacZ:: Tn 5 ) has been described previously [39].…”

“…The PCR fragment was digested with Eco RI and Bam HI and ligated into plasmid pUHE23‐2 [18], previously digested with the same two endonucleases and treated with alkaline phosphatase to prevent self‐ligation. The ligated mixture was transformed into host E. coli strain NF1830, which carries an F′lacI q1 episome and overproduces the lac‐repressor so that transcription of the cloned gene is repressed until induction by isopropyl thio‐β‐ d ‐galactoside [39], and plated on Luria–Bertani agar plates supplemented with ampicillin (100 µg·mL −1 ). The selected plasmid (pLSF2) contained the upp gene and the sequence was found to be identical to the sequence of the published sequence of the upp gene of S. solfataricus , the open reading frame SSO0231 [16].…”

Structure of the dimeric form of CTP synthase fromSulfolobus solfataricus

“…By use of the BamHI and SalI restriction endonuclease sites introduced into the PCR product by sspyrg1 and sspyrg2, respectively, and indicated in italics in the above sequences, an expression vector was constructed by cloning the S. solfataricus pyrG PCR fragment into the E. coli DNA vector pUHE23-2 (Deuschle et al, 1986), resulting in the plasmid pKDS4. CTP synthase was synthesized by growing E. coli strain NF1830 (Andersen et al, 1992) transformed with pKDS4 with vigorous shaking at 310 K in a chemically defined basic salt medium (Clark & Maaløe, 1967) supplied with 10 g tryptone, 5 g yeast extract, 1 g glucose and 100 mg ampicillin per litre. Isopropyl -d-1-thiogalactopyranoside (0.5 mM) was added when the OD 436 was equal to 0.5.…”

Controlling Messenger RNA Stability in Bacteria: Strategies for Engineering Gene Expression

“…By imparting different stabilities on each gene in a polycistronic mRNA, the cell has an additional means of manipulating the protein levels from a single promoter. Operons containing differential internal stabilities within a polycistronic mRNA are found in a wide range of prokaryotes and include the E. coli unc ( atp ), melibiose , and rph‐pyrE operons, the Z. mobilis glf‐zwf‐glk operon, and the S. typhimurium histidine transport system (Alifano et al, 1992; Andersen et al, 1992; Liu et al, 1992; McCarthy et al, 1991; Mejia et al, 1992; Patel and Dunn, 1995; Romeo and Zusman, 1992; Shimamato et al, 1994). In addition to single operons, the regulatory role of mRNA stability has been implicated in multigene metabolic pathways such as the Z. mobilis glycolytic and fermentative pathways (Mejia et al, 1992).…”