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PubMed1985

Regulation of aspartate transcarbamoylase synthesis in Escherichia coli: analysis of deletion mutations in the promoter region of the pyrBI operon.

The catalytic and regulatory polypeptide chains of Escherichia coli aspartate transcarbamoylase are encoded by the pyrB and pyrI genes, respectively, which constitute a single transcriptional unit in the pyrBI operon. The DNA sequence immediately preceding the first structural gene, pyrB, contains a short open reading frame that could encode a 44-amino acid leader peptide and a (G+C)-rich region of dyad symmetry followed by eight thymidine residues. Synthesis of the enzyme is negatively controlled at the level of transcription depending on the cellular level of UTP, and an attenuation mechanism has been proposed to account for the 70-fold increase in pyrBI expression on pyrimidine starvation. The potential role of the dyad and eight thymidines as an attenuator was tested with a plasmid containing the promoter region of the pyrBI operon upstream of the galK coding sequence. When cells containing this plasmid, pPYRB10, were grown in a medium low in uracil, there was an 83-fold increase in galactokinase activity compared with the same cells grown at high uracil levels. This regulation is similar to that for aspartate transcarbamoylase synthesis in cells depleted of pyrimidines. Deletions constructed in the promoter region of pPYRB10 from the 3' side produced one plasmid that retained normal control of galK expression and five that exhibited greatly reduced regulation. Nucleotide sequence determination showed that the one deletion mutation that was functionally similar to the wild-type plasmid contained the entire region of dyad symmetry, including the eight thymidines. The plasmids with more extensive deletions lacked the region with dyad symmetry and the eight thymidines. One of the deletion mutants that exhibited very low levels of regulation lacks the entire sequence coding for the putative leader peptide up to the major promoter. The results demonstrating the crucial role of a 19-nucleotide sequence (from -33 to -15) support an attenuation model but indicate that other mechanisms also contribute to the regulation of the pyrBI operon.

Aspartate Carbamoyltransferase↗

PubMed1987

Autogenous control of the S10 ribosomal protein operon of Escherichia coli: genetic dissection of transcriptional and posttranscriptional regulation.

The S10 ribosomal protein operon is regulated autogenously by the product of one of the genes of the operon, the gene encoding ribosomal protein L4. We have used site-directed mutagenesis to isolate leader mutations affecting L4 control. The phenotypes of these mutants demonstrate that L4 regulates both transcription and translation of the S10 operon. Several mutations abolish both levels of L4 control; others eliminate either transcriptional or translational control with little or no effect on the other mode of regulation. We conclude that L4-mediated transcriptional and translational control share some sequence requirements, but the two regulatory processes recognize somewhat different features of the S10 leader. Primary as well as secondary structures within the S10 leader appear to be involved.

Escherichia coli↗

PubMed1987

his operons of Escherichia coli and Salmonella typhimurium are regulated by DNA supercoiling.

The hisW mutations of Salmonella typhimurium are highly pleiotropic mutations that elevate his operon expression, reduce ilv gene expression, alter stable RNA metabolism, and confer defective growth properties. The hisW mutations are highly linked to a naladixic acid-resistant gyrA mutation of S. typhimurium. Multicopy recombinant plasmids containing the Escherichia coli gyrA gene are able to complement both the growth defects and the elevated his operon expression associated with the hisW mutations. We conclude that hisW mutations are alleles of the gyrA gene. The hisU1820 mutant of S. typhimurium exhibits many of the same phenotypes as hisW mutants. Several lines of evidence, including high transduction linkage to recF, suggest that hisU1820 is an allele of gyrB. Finally, well-characterized gyrA and gyrB alleles of E. coli are also his regulatory mutations. We propose that a wild-type degree of chromosomal superhelicity is required for maximal production of histidyl-tRNA and normal his operon regulation.

Alcohol Oxidoreductases↗

PubMed1992

Leucine-responsive regulatory protein controls the expression of both the pap and fan pili operons in Escherichia coli.

The methylation blocking factor gene (mbf) in Escherichia coli is required for specific methylation inhibition of two DNA GATC sites upstream of the papBA pilin promoter and transcriptional activation of pap. Complementation and mutational analysis using pap-lac and ilvIH-lac operon fusions indicates that the mbf gene is identical to a recently described global regulatory gene lrp (leucine-responsive regulatory protein) that acts as a positive regulator of some genes and a negative regulator of others in E. coli. DNA sequence analysis of an mbf::mTn10 insertion showed that the mbfDNA sequence was identical to lrp. Thus Lrp inhibits DNA methylation at specific GATC sites. We also show that Lrp positively regulates transcription of the fan operon, which encodes K99 pili of diarrheagenic E. coli. Purified Lrp was found to bind to DNA fragments encompassing the pap and fan promoters, which is consistent with previous results indicating that Lrp controls gene expression by binding to regulatory DNA sites. Exogenous leucine significantly reduced fan transcription and K99 pili expression, similar to results obtained with the ilvIH operon. However, pap gene expression was unresponsive to leucine, which distinguishes pap from other lrp-regulated genes whose expression is modulated by leucine.

Amino Acid Sequence↗

PubMed1992

A transcriptional silencer downstream of the promoter in the osmotically controlled proU operon of Salmonella typhimurium.

The proU operon of Salmonella typhimurium is induced by conditions of high osmolality. The cis-acting sequences that mediate osmotic control of transcription were characterized by deletion analysis. The nucleotide sequence between -60 and +274 (relative to the transcription start point) is sufficient for normal osmotic control. Deletions that removed sequences upstream of position +274 but left the promoter intact resulted in greatly increased expression from the proU promoter in the absence of osmotic stress. Thus, the transcription control region of the proU operon consists of two discrete components: (i) the promoter and (ii) a negatively acting site that overlaps the coding sequence of the first structural gene of the operon, proV. That this negative regulatory element is a transcriptional terminator or mRNA processing site was ruled out. Our results suggest that the negative regulatory element behaves as a transcriptional silencer that inhibits transcription initiation at the proU promoter in medium of low osmolality by some action at a distance. We propose several possible mechanisms for the function of this regulatory site.

Base Sequence↗

PubMed1994

KorA protein of promiscuous plasmid RK2 controls a transcriptional switch between divergent operons for plasmid replication and conjugative transfer.

The trfA and trb operons, encoding genes essential for replication and conjugative transfer of broad host range plasmid RK2, are transcribed divergently. Deletion analysis presented here indicates that trfAp and trbAp are arranged as face to face promoters. The presence of the korA gene, whose product is known to repress seven operons on RK2, including the trfA operon, is shown here to stimulate trbAp. The effect of korA on trbAp is mimicked by the trfAp-1 promoter down mutation, suggesting that a reduction in the activity of trfAp is required for derepression of trbAp activity. The trfAp-1 mutation reduces RNA polymerase binding and open complex formation at trfAp but does not stimulate melting at trbAp in vitro. Therefore, the inhibition of trbAp is most probably due to forward transcription initiated at trfAp. The simultaneous inhibition/stimulation by KorA is seen even in the presence of the other repressors KorB and TrbA, which act at this region, thus providing a dominant mode of coordinating plasmid replication and transfer. This may be one of the keys to understanding how the maintenance and spread of promiscuous plasmids are balanced in different environments.

Amino Acid Sequence↗

PubMed1994

Role of an Escherichia coli stress-response operon in stationary-phase survival.

The phage shock protein operon (pspABCE) of Escherichia coli is strongly expressed in response to stressful environmental conditions, such as heat shock, ethanol treatment, osmotic shock, and filamentous phage infection. We show that bacteria lacking the pspABC genes exhibit a substantial decrease in the ability to survive prolonged incubation in stationary phase under alkaline conditions (pH 9). The psp mutant bacteria grow approximately as well as wild-type strains in the alkaline medium, and stationary-phase survival of the psp mutants improves substantially at pH values closer to the optimal growth range (pH 6-8). In late stationary-phase (1- to 2-day-old) cells, the operon can be strongly induced under certain conditions, and PspA can become one of the most highly expressed bacterial proteins. The combination of stationary-phase starvation and alkaline pH is likely to place a severe strain on the maintenance of endogenous energy sources, and, consistent with these effects, we find that psp expression is also induced by uncouplers of oxidative phosphorylation and other agents that interfere with energy production. The death rate of psp mutants in stationary phase is accelerated by the presence of wild-type bacteria in the same culture, suggesting that the psp operon may play a significant role in enabling E. coli to compete for survival under nutrient- or energy-limited conditions.

Bacterial Proteins↗

PubMed1995

Identification of the Bacillus subtilis pur operon repressor.

Transcription of the Bacillus subtilis pur operon is repressed in response to a signal of excess adenine. We have purified the repressor protein and have identified, cloned, and overexpressed the purR regulatory gene that controls transcription initiation of the operon. B. subtilis purR encodes a 62-kDa homodimer that binds to the pur operon control region. The PurR binding site which overlaps the promoter encompasses approximately 110 bp. The protein-DNA interaction is inhibited by 5-phosphoribosyl 1-pyrophosphate. A mutation that deletes the repressor binding site or one that disrupts purR abolishes binding activity in vitro and repression of transcription in vivo in response to the excess adenine signal. These results lead to a model in which an excess-adenine signal is transmitted to PurR via the 5-phosphoribosyl 1-pyrophosphate pool. In addition, purR is autoregulated. There is no structural or mechanistic similarity between the B. subtilis and Escherichia coli purine repressors.

Amino Acid Sequence↗

PubMed1996

The lpf fimbrial operon mediates adhesion of Salmonella typhimurium to murine Peyer's patches.

We investigated the role of the Salmonella typhimurium fimbrial operon formed by the genes lpfABCDE in infection of mice. A mutant in lpfC, the gene encoding the fimbrial outer membrane usher, had an approximately 5-fold increased 50% lethal dose when administered orally to mice. When mice were infected with a mixture of the lpfC mutant and isogenic wild-type S. typhimurium, the lpfC mutant was recovered in lower numbers from Peyer's patches, mesenteric lymph nodes, liver, and spleen. In an organ culture model using murine intestinal loops, lpfC mutants were shown to be associated in lower numbers than wild-type bacteria with Peyer's patches but not with villous intestine. The defect of the lpfC mutant in adhesion to Peyer's patches could be complemented by introducing lpfABCDE on a cosmid. Similarly, heterologous expression of the Salmonella lpf operon in Escherichia coli resulted in an increased adhesion to histological thin sections of Peyer's patch lymph follicles. Electron microscopic analysis of histological sections taken from Peyer's patches after intragastric infection of mice showed that, in contrast to the S. typhimurium wild type, the isogenic lpfC mutant did not destroy M cells of the follicle-associated epithelium. These data show that the Salmonella lpf operon is involved in adhesion to murine Peyer's patches.

Animals↗

PubMed1997

Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis.

The genomes of most eukaryotes are composed of genes arranged on the chromosomes without regard to function, with each gene transcribed from a promoter at its 5' end. However, the genome of the free-living nematode Caenorhabditis elegans contains numerous polycistronic clusters similar to bacterial operons in which the genes are transcribed sequentially from a single promoter at the 5' end of the cluster. The resulting polycistronic pre-mRNAs are processed into monocistronic mRNAs by conventional 3' end formation, cleavage, and polyadenylation, accompanied by trans-splicing with a specialized spliced leader (SL), SL2. To determine whether this mode of gene organization and expression, apparently unique among the animals, occurs in other species, we have investigated genes in a distantly related free-living rhabditid nematode in the genus Dolichorhabditis (strain CEW1). We have identified both SL1 and SL2 RNAs in this species. In addition, we have sequenced a Dolichorhabditis genomic region containing a gene cluster with all of the characteristics of the C. elegans operons. We show that the downstream gene is trans-spliced to SL2. We also present evidence that suggests that these two genes are also clustered in the C. elegans and Caenorhabditis briggsae genomes. Thus, it appears that the arrangement of genes in operons pre-dates the divergence of the genus Caenorhabditis from the other genera in the family Rhabditidae, and may be more widespread than is currently appreciated.

Animals↗

PubMed1998

Two functionally dependent acetylcholine subunits are encoded in a single Caenorhabditis elegans operon.

The deg-3 gene from the nematode Caenorhabditis elegans encodes an alpha subunit of a nicotinic acetylcholine receptor that was first identified by a dominant allele, u662, which produced neuronal degeneration. Because deg-3 cDNAs contain the SL2 trans-spliced leader, we suggested that deg-3 was transcribed as part of a C. elegans operon. Here we show that des-2, a gene in which mutations suppress deg-3(u662), is the upstream gene in that operon. The des-2 gene also encodes an alpha subunit of a nicotinic acetylcholine receptor. As expected for genes whose mRNAs are formed from a single transcript, both genes have similar expression patterns. This coexpression is functionally important because (i) des-2 is needed for the deg-3(u662) degenerations in vivo; (ii) an acetylcholine-gated channel is formed in Xenopus oocytes when both subunits are expressed but not when either is expressed alone; and (iii) channel activity, albeit apparently altered from that of the wild-type channel, results from the expression of a u662-type mutant subunit but, again, only when the wild-type DES-2 subunit is present. Thus, the operon structure appears to regulate the coordinate expression of two channel subunits.

Acetylcholine↗

PubMed1999

Transcriptional coupling between the divergent promoters of a prototypic LysR-type regulatory system, the ilvYC operon of Escherichia coli.

The twin-domain model [Liu, L. F. & Wang, J. C. (1987) Proc. Natl. Acad. Sci. USA 84, 7024-7027] suggests that closely spaced, divergent, superhelically sensitive promoters can affect the transcriptional activity of one another by transcriptionally induced negative DNA supercoiling generated in the divergent promoter region. This gene arrangement is observed for many LysR-type-regulated operons in bacteria. We have examined the effects of divergent transcription in the prototypic LysR-type system, the ilvYC operon of Escherichia coli. Double-reporter constructs with the lacZ gene under transcriptional control of the ilvC promoter and the galK gene under control of the divergent ilvY promoter were used to demonstrate that a down-promoter mutation in the ilvY promoter severely decreases in vivo transcription from the ilvC promoter. However, a down-promoter mutation in the ilvC promoter only slightly affects transcription from the ilvY promoter. In vitro transcription assays with DNA topoisomers showed that transcription from the ilvC promoter increases over the entire range of physiological superhelical densities, whereas transcription initiation from the ilvY promoter exhibits a broad optimum at a midphysiological superhelical density. Evidence that this promoter coupling is DNA supercoiling-dependent is provided by the observation that a novobiocin-induced decrease in global negative superhelicity results in an increase in ilvY promoter activity and a decrease in ilvC promoter activity predicted by the in vitro data. We suggest that this transcriptional coupling is important for coordinating basal level expression of the ilvYC operon with the nutritional and environmental conditions of cell growth.

Bacterial Proteins↗

PubMed1995

Proline dehydrogenase activity of the transcriptional repressor PutA is required for induction of the put operon by proline.

The proline utilization (put) operon from Salmonella typhimurium consists of the putP gene, encoding a proline transporter, and the putA gene, encoding an enzyme with both proline dehydrogenase and 1-pyrroline-5-carboxylate dehydrogenase activities. In addition to these two enzymatic activities, the PutA protein is a transcriptional repressor that regulates the expression of putP and putA in response to the availability of proline. We report the isolation of super-repressor mutants of PutA that decrease expression from the putA promoter in the presence or absence of proline. None of the mutants exhibited increased affinity for the DNA in the put regulatory region in vitro. Although DNA binding by wild-type PutA was prevented by the addition of proline and an artificial electron acceptor, DNA binding by the two strongest super-repressors was not prevented under identical conditions. The proline dehydrogenase activity of the purified mutant proteins showed altered kinetic properties (increased Km(Pro), reduced Vmax, or a completely null phenotype). The observation that these mutations simultaneously affect induction by proline and proline dehydrogenase activity suggests that a single proline-binding site is involved in both proline dehydrogenase activity and induction of the expression of the put operon. Furthermore, the results indicate that the proline dehydrogenase activity of PutA is essential for induction of the put operon by proline.

Alleles↗

PubMed1995

Identification of two new genes in the Pseudomonas aeruginosa amidase operon, encoding an ATPase (AmiB) and a putative integral membrane protein (AmiS).

The nucleotide sequence of the amidase operon of Pseudomonas aeruginosa has been completed and two new genes identified amiB and amiS. The complete gene order for the operon is thus amiEBCRS. The amiB gene encodes a 42-kDa protein containing an ATP binding motif that shares extensive homology with the Clp family of proteins and also to an open reading frame adjacent to the amidase gene from Rhodococcus erythropolis. Deletion of the amiB gene has no apparent effect on inducible amidase expression and it is thus unlikely to encode a regulatory protein. A maltose-binding protein-AmiB fusion has been purified and shown to have an intrinsic ATPase activity (Km = 174 +/- 15 mM; Vmax = 2.4 +/- 0.1 mM/min/mg), which is effectively inhibited by ammonium vanadate and ADP. The amiS gene encodes an 18-kDa protein with a high content of hydrophobic residues. Hydropathy analysis suggests the presence of six transmembrane helices in this protein. The AmiS sequences is homologous to an open reading frame identified adjacent to the amidase gene from Mycobacterium smegmatis and to the ureI gene from the urease operon of Helicobacter pylori. AmiS and its homologs appear to be a novel family of integral membrane proteins. Together AmiB and AmiS resemble two components of an ABC transporter system.

Adenosine Triphosphatases↗

PubMed1996

Intracellular Na+ regulates transcription of the ntp operon encoding a vacuolar-type Na+-translocating ATPase in Enterococcus hirae.

The Gram-positive bacterium Enterococcus hirae has a vacuolar-type Na+-translocating ATPase that is encoded by the ntp operon (ntpFIKECGABDHJ) (Takase, K., Kakinuma, S., Yamato, I., Konishi, K., Igarashi, K., and Kakinuma, Y. (1994) J. Biol. Chem. 269, 11037-11044). Primer extension experiments identified the start site of transcription of this operon upstream of the ntpF gene. In parallel with the increases of both Na+-pumping activity in whole cells and Na+-stimulated ATPase activity in the membranes, the amounts of the two major subunits (A and B) of this enzyme increased remarkably in cells grown on medium containing high concentrations of NaCl but not on medium containing KCl or sorbitol. Chloramphenicol completely abolished the increases of the enzyme activity and the amounts of A and B subunits, suggesting that the Na+-ATPase level increased by de novo synthesis of the enzyme with the stimulation of high concentrations of the external sodium ions. Finally, Western blot and Northern blot experiments revealed that the increase in the Na+-ATPase level with the external Na+ was further accelerated by addition of an ionophore, such as monensin, which rendered the cell membrane permeable to Na+. These results suggest that the transcription of the Na+-ATPase operon is regulated by the intracellular concentration of sodium ions.

Adenosine Triphosphatases↗

PubMed1997

DNA topoisomerases regulate R-loop formation during transcription of the rrnB operon in Escherichia coli.

Recent in vivo and in vitro studies have suggested an important role for DNA topoisomerases in regulating R-loop formation during transcription in Escherichia coli. In the present report we present genetic and biochemical evidence strongly suggesting that R-loop formation can occur during transcription of a portion of the rrnB operon and that it is regulated by DNA topoisomerase activity. We found that a multicopy plasmid (pBR322) carrying an heavily transcribed portion of the rrnB operon cannot be transformed in topA mutants unless RNase H is overproduced. Transcription of the 567-base pair HindIII fragment from the rrnB operon allows the extraction of large amount of R-looped plasmid DNAs from a topA mutant, in a manner that depends on the intracellular level of RNase H activity. When DNA gyrase is sufficiently active, hypernegatively supercoiled plasmid DNA is produced if the same DNA fragment is transcribed in a topA mutant. The formation of such topoisomers most likely reflect the presence of extensive R-loops since it is sensitive to the intracellular level of RNase H activity. Finally, the formation of R-looped plasmid DNAs in an in vitro transcription system using phage RNA polymerases is also detected when the 567-base pair HindIII fragment is transcribed on a negatively supercoiled DNA template.

DNA Topoisomerases, Type I↗

PubMed1997

Organ-specific transcription of the rrn operon in spinach plastids.

The spinach rrn operon is used as a model system to study transcriptional regulation in higher plant photosynthetic and non-photosynthetic plastids. We performed capping experiments to determine whether P1, PC, or P2 promoters are employed for rrn transcription start sites in cotyledon and root tissues. By using a new method of analysis of capped RNA we demonstrate for the first time that 1) in both organs the rrn operon is expressed in a constitutive manner by cotranscription with the preceding tRNA(GAC)Val gene, and 2) the PC transcription start site is used only in cotyledons and leaves, i.e. we demonstrate the organ-specific usage of a plastid promoter. Both start sites, PC and that of the tRNA(GAC)Val cotranscript, lack Escherichia coli-like consensus sequences. The cotranscript is initiated 457 base pairs upstream of the tRNA(GAC)Val gene. The PC-specific DNA-binding factor, CDF2, is not detectable in root tissues confirming its regulatory role in PC-initiated rrn expression and the organ specificity of PC expression. Furthermore, our results show that rrn operon expression patterns differ in spinach and tobacco indicating species-specific transcriptional regulation of plant plastid gene expression.

Base Sequence↗

PubMed2000

Reproducing tna operon regulation in vitro in an S-30 system. Tryptophan induction inhibits cleavage of TnaC peptidyl-tRNA.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Catabolite repression regulates transcription initiation, whereas excess tryptophan induces antitermination at Rho factor-dependent termination sites in the leader region of the operon. Synthesis of the leader peptide, TnaC, is essential for antitermination. BoxA and rut sites in the immediate vicinity of the tnaC stop codon are required for termination. In this paper we use an in vitro S-30 cell-free system to analyze the features of tna operon regulation. We show that transcription initiation is cyclic AMP (cAMP)-dependent and is not influenced by tryptophan. Continuation of transcription beyond the leader region requires the presence of inducing levels of tryptophan and synthesis of the TnaC leader peptide. Using a tnaA'-'trpE fusion, we demonstrate that induction results in a 15-20-fold increase in synthesis of the tryptophan-free TnaA-TrpE fusion protein. Replacing Trp codon 12 of tnaC by an Arg codon, or changing the tnaC start codon to a stop codon, eliminates induction. Addition of bicyclomycin, a specific inhibitor of Rho factor action, substantially increases basal level expression. Analyses of tna mRNA synthesis in vitro demonstrate that, in the absence of inducer transcription is terminated and the terminated transcripts are degraded. In the presence of inducer, antitermination increases the synthesis of the read-through transcript. TnaC synthesis is observed in the cell-free system. However, in the presence of tryptophan, a peptidyl-tRNA also appears, TnaC-tRNA(Pro). Our findings suggest that inducer acts by preventing cleavage of TnaC peptidyl-tRNA. The ribosome associated with this newly synthesized peptidyl-tRNA presumably stalls at the tnaC stop codon, blocking Rho's access to the BoxA and rut sites, thereby preventing termination. 1-Methyltryptophan also is an effective inducer in vitro. This tryptophan analog is not incorporated into TnaC.

Bacterial Proteins↗