RNA polymerase involvement in the regulation of expression of Salmonella typhimurium pyr genes. Isolation and characterization of a fluorouracil-resistant mutant with high, constitutive expression of the pyrB and pyrE genes due to a mutation in rpoBC.

Jensen, Kaj Frank; Neuhard, Jan; Schack, Lise

doi:10.1002/j.1460-2075.1982.tb01126.x

Cited by 57 publications

(35 citation statements)

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“…The receptor which senses these regulatory signals seems to be the elongating RNA polymerase. First, the only type of mutants identified which overproduce the pyrimidine biosynthetic enzymes, while at the same time containing high intracellular pools of repressing nucleotides, are rpoBC mutants [7]. Second, the purified RNA polymerase of one such mutant was found to have a 4-6-fold elevated K,,, for UTP in the elongation of transcripts of T7 DNA and synthetic DNAs (K. F. Jensen, unpuplished observations).…”

Section: Discussionmentioning

confidence: 99%

Nucleotide sequence of the Escherichia coli pyrE gene and of the DNA in front of the protein-coding region

et al. 1983

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Orotate phosphoribosyltransferase (EC 2.4.2.10) was purified to electrophoretic homogeneity from a strain of Escherichia coli containing t h e p y r E gene cloned o n a multicopy plasmid. The relative molecular masses (M,) of the native enzyme and its subunit were estimated by means of gel filtration and electrophoresis in the presence of dodecyl sulfate. The amino acid sequences at the N and C termini, as well as the amino acid composition, were determined.The nucleotide sequence of the structuralpyrE gene, including 394 nucleotide residues preceding the beginning of the coding frame, was also established. From the results the following conclusions may be drawn.Orotate phosphoribosyltransferase is a dimeric protein with subunits of M, 23 326 consisting of 21 1 amino acid residues. The pyrE gene is transcribed in a counter-clockwise direction from the E. coli chromosome as an mRNA with a considerable leader segment in front of the protein-coding region. This leader contains a structure with features characteristic for a (translated ?) rho-independent transcriptional terminator, which is preceded by a cluster of uridylate residues. This indicates that the frequency ofpyrE transcription is regulated by an R N A polymerase (UTP) modulated attenuation Orotate phosphoribosyltransferase (EC 2.4.2.10) catalyses one of the reactions in the de nova synthesis of UMP (Fig. 1). The pyr genes are scattered on the chromosome of enteric bacteria, and the structural gene for orotate phosphoribosyltransferase, pyrE, is located at 81 min o n the Eschrrichia coli linkage map, between dut and spoT [I].Both in E. coli and in Sulmonella typhimuriurn the expression of p y B , pyrE, and p y r F is repressed by a uridine nucleotide, which is U T P o r UDP [2-41, while it is elevated by partial guanine starvation [ 5 , 61. True regulatory mutants defective in the control of pyr gene activity have not been identified. However, it has been found that some R N A polymerase mutants (rpoBC) display high, constitutive pyrB and pyrE expression, while at the same time they contain high intracellular concentrations of uridine nucleotides 171. All other identified mutants with enhanced expression of the pyr genes have been shown to harbor defects in nucleotide interconversion pathways (pyrH, guaB) [S, 81. These observations indicate that R N A polymerase is involved in sensing the regulatory signals in pyr gene regulation. To characterize the biochemistry behind the control of pyrimidine biosynthesis further we have initiated a study of the individual pyr genes. This paper describes the nucleotide sequence of the E. coli p y r E gene and of its regulatory region.This gene had previously been cloned onto various multicopy plasmids [9 -111, and from a strain containing one of these [I03 we have purified the gene product, orotate phosphoribosyltransferase, and characterized its structure in some detail. The same plasmid served as a source of D N A restriction fragments for D N A sequencing. The results indicate that t h e p y r E gene is _ _ __ Ahhr...

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Section: Discussionmentioning

confidence: 99%

Nucleotide sequence of the Escherichia coli pyrE gene and of the DNA in front of the protein-coding region

et al. 1983

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“…⌬relA derivatives of strains KT2244 and KT2246 were constructed by transduction of strains KP1469 and KP1475 to kanamycin resistance as above. Four independent ⌬relA isolates of KP1475 were screened on L broth plates to verify the absence of fast-growing revertants of the slow-growth phenotype on rich media conferred by the rpoBC allele (26). The lrp-1::Tn5 mutants KT2402, KT2404, and KT2406 were constructed by P22 transduction of strains LT2, KT2282, and KT2354, respectively, to kanamycin resistance using lysates prepared on strain GA446 followed by screening for serine sensitivity at 42°C (Lrp Ϫ phenotype [40]).…”

Section: Methodsmentioning

confidence: 99%

“…It was reasoned that it should be possible to compensate for the reduced basal ppGpp levels after downshifts of a relA mutant in a strain harboring an RNA polymerase with a reduced RNA chain elongation rate without resorting to pyrimidine limitations. To test this idea, the ⌬relA allele was introduced into a strain of S. enterica serovar Typhimurium LT2, KP1475, expressing an RNA polymerase with a reduced RNA chain elongation rate (26,27). As seen in Table 4, the ⌬relA derivative of the parent strain, KT2244, showed recovery defects similar to the other S. enterica serovar Typhimurium LT2 ⌬relA derivatives, but in all cases the defects were reversed in the presence of both leucine and valine.…”

Section: Vol 183 2001 Ppgpp and Regulation Of Gene Expression 6187mentioning

confidence: 99%

Comparison of Δ relA Strains of Escherichia coli and Salmonella enterica Serovar Typhimurium Suggests a Role for ppGpp in Attenuation Regulation of Branched-Chain Amino Acid Biosynthesis

Tedin

Norel

2001

J Bacteriol

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The growth recovery of Escherichia coli K-12 and Salmonella enterica serovar Typhimurium ⌬relA mutants were compared after nutritional downshifts requiring derepression of the branched-chain amino acid pathways. Because wild-type E. coli K-12 and S. enterica serovar Typhimurium LT2 strains are defective in the expression of the genes encoding the branch point acetohydroxy acid synthetase II (ilvGM) and III (ilvIH) isozymes, respectively, ⌬relA derivatives corrected for these mutations were also examined. Results indicate that reduced expression of the known global regulatory factors involved in branched-chain amino acid biosynthesis cannot completely explain the observed growth recovery defects of the ⌬relA strains. In the E. coli K-12 MG1655 ⌬relA background, correction of the preexisting rph-1 allele which causes pyrimidine limitations resulted in complete loss of growth recovery. S. enterica serovar Typhimurium LT2 ⌬relA strains were fully complemented by elevated basal ppGpp levels in an S. enterica serovar Typhimurium LT2 ⌬relA spoT1 mutant or in a strain harboring an RNA polymerase mutation conferring a reduced RNA chain elongation rate. The results are best explained by a dependence on the basal levels of ppGpp, which are determined by relAdependent changes in tRNA synthesis resulting from amino acid starvations. Expression of the branched-chain amino acid operons is suggested to require changes in the RNA chain elongation rate of the RNA polymerase, which can be achieved either by elevation of the basal ppGpp levels or, in the case of the E. coli K-12 MG1655 strain, through pyrimidine limitations which partially compensate for reduced ppGpp levels. Roles for ppGpp in branched-chain amino acid biosynthesis are discussed in terms of effects on the synthesis of known global regulatory proteins and current models for the control of global RNA synthesis by ppGpp.

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“…They are located in the distal end of rpoB or in rpoC (14). One of the mutants, KP1475, displays a 100-fold increase in pyrB expression; the other two show only moderately enhanced levels of enzyme coded for by pyrB.…”

Section: Methodsmentioning

confidence: 99%

Isolation of Escherichia coli rpoB mutants resistant to killing by lambda cII protein and altered in pyrE gene attenuation

Hammer¹,

Jensen²,

Poulsen³

et al. 1987

J Bacteriol

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Escherichia coli mutants simultaneously resistant to rifampin and to the lethal effects of bacteriophage A clI protein were isolated. The sck mutant strains carry alterations in rpoB that allow them to survive cII killing (thus the name sck), but that do not impair either the expression of cII or the activation by cII of the A promoters PE and pl. The sck-1, sck-2, and sck-3 mutations modify transcription termination. The growth of X, but not of the N-independent A variant, A nin-5, is hindered by these mutations, which act either alone or in concert with the bacterial nusAl mutation. In contrast to their effect on k growth, the three mutations reduce transcription termination in bacterial operons. The E. coli pyrE gene, which is normally regulated by attenuation, is expressed constitutively in the mutant strains. The sck mutations appear to prevent pyrE attenuation by slowing the rate of transcriptional elongation of the pyrE leader sequence. The sck-6 mutation, unlike the other sck mutations, neither increases pyrE expression nor inhibits the ability of k to suppress transcription termination. Instead, the sck-6 mutation blocks the growth of the A variants A nin-5 and K red-3.The cII protein of bacteriophage lambda plays both positive and negative roles in viral development. It stimulates transcription initiation from the phage Pl, PE, and PQ promoters (9,21,22). The activation of PE and PQ inhibits the expression of early and late viral lytic genes; presumably, transcription initiating at these promoters converges with and inhibits transcription from the lytic A PR promoter.Expression of clI from a multicopy plasmid is lethal to Escherichia coli (28). This lethality may result from a severe depression in host protein synthesis observed after clI induction. We assumed that the initial reaction in cIIinduced killing was an interaction between RNA polymerase and the cIT protein at certain bacterial promoters. Convergent transcription from these promoters might depress the expression of vital bacterial genes. By analogy with mutations in rpoB (the d subunit of RNA polymerase) which block the transcription antitermination activity of the X N gene product (8), we sought rpoB mutants which survived cII killing (sck mutants). The properties of four such sck mutants are described below. Although we expected these rpoB mutations to block the action of the cIT product, our results indicate that the mutant polymerases still, in fact, interact with the cII protein. Instead of affecting the action of the cII product, some of the mutations appear to affect transcription termination; they display or enhance the Nusphenotype and derepress the bacterial pyrE gene, which is normally attenuation regulated. MATERIALS AND METHODSMedia. LB medium has been described (19 N6017 (21) carries a cI857 Nam7 Nam53 int2 xisl prophage with X DNA deleted from between the SalI and XhoI sites at X coordinates 32745 and 33498, respectively; it lacks cIII, kil, gam, and bet. In addition, the Hi deletion removes all prophage genes from cro to attR...

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RNA polymerase involvement in the regulation of expression of Salmonella typhimurium pyr genes. Isolation and characterization of a fluorouracil-resistant mutant with high, constitutive expression of the pyrB and pyrE genes due to a mutation in rpoBC.

Cited by 57 publications

References 32 publications

Nucleotide sequence of the Escherichia coli pyrE gene and of the DNA in front of the protein-coding region

Nucleotide sequence of the Escherichia coli pyrE gene and of the DNA in front of the protein-coding region

Comparison of Δ relA Strains of Escherichia coli and Salmonella enterica Serovar Typhimurium Suggests a Role for ppGpp in Attenuation Regulation of Branched-Chain Amino Acid Biosynthesis

Isolation of Escherichia coli rpoB mutants resistant to killing by lambda cII protein and altered in pyrE gene attenuation

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