Enhancement of RNA polymerase binding to promoters by a transcriptional activator, OmpR, in Escherichia coli: its positive and negative effects on transcription.

Tsung, Kangla; Brissette, Renée; Inouye, Masayori

doi:10.1073/pnas.87.15.5940

Cited by 29 publications

(13 citation statements)

References 23 publications

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“…In the cell, many variables presumably affect which step is rate-limiting, including temperature, DNA superhelicity, ionic strength, nutrient -conditions, the concentration of free RNA polymerase, mutations, and the binding of transcription factors (13,16,(18)(19)(20)(21)(22). Based upon the results presented here, any variable that changes the rate-limiting step in transcription of a specific promoter could potentially transform a repressor into an activator or vice versa.…”

Section: Discussionmentioning

confidence: 99%

Dual regulation of open-complex formation and promoter clearance by Arc explains a novel repressor to activator switch.

Smith

Sauer

1996

Proc. Natl. Acad. Sci. U.S.A.

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In studies of variants of the Pant promoter of bacteriophage P22, the Arc protein was found not only to slow the rate at which RNA polymerase forms open complexes but also to accelerate the rate at which the enzyme clears the promoter. These dual activities permit Arc, bound at a single operator subsite, to act as an activator or as a repressor of different promoter variants. For During late lytic growth of bacteriophage P22, Arc represses transcription from the Pant and Pmnt promoters in the immunity I operon (2, 3). This activity of Arc is mediated by binding to tandem subsites of the arc operator, positioned between the -10 and -35 promoter elements (3). Each operator subsite binds a single Arc dimer, and cooperative interactions between tandemly bound dimers contribute to stabilization of the protein-operator complex (4, 5). Here, we present evidence that Arc affects multiple steps in transcription initiation at Pant. Using variants of the Pant promoter, we show that Arc both slows open-complex formation and accelerates promoter clearance. These dual and opposing activities permit Arc to act as an activator or as a repressor, depending upon the intrinsic rates of different initiation steps for a given promoter and how Arc changes these rates. An Arc dimer bound at the operator subsite next to the -35 promoter element of Pant is both necessary and sufficient for the acceleration of promoter clearance. This positive activity is also diminished by the RA23 mutation of Arc which removes a phosphate-backbone contact in the protein-operator complex (5, 6). MATERIALS AND METHODSProteins. Arc and variants of Arc contained the st6 or stll C-terminal extensions that minimize proteolysis in vivo and allow purification by nickel-affinity chromatography (7). Neither addition affects the activity of Arc (7) To assay open-complex formation, RNA polymerase (7.5 nM) was added to promoter DNA (0.2 nM), and the reaction was quenched after different times by the addition of heparin to 100 ,ug/ml and sucrose to 5%. Bound and free DNA fragments were resolved on 4% polyacrylamide, 0.5x TBE gels.To assay promoter clearance by footprinting, RNA polymerase and promoter DNA were allowed to form open complexes, the reactions were diluted 50-fold into buffer containing 0.1 ,ug/ml heparin with or without Arc-SL35, and NTPs were added to initiate transcription. At different times, I aliquots were removed and added to DNAse I (final concentration of 18 ng/ml) for 60 s, and cleavage was quenched by addition of an equal volume of 2.5 M ammonium acetate, 20 mM Na2EDTA, and 10 u.g/ml salmon sperm DNA. Digestion products were analyzed on 6% polyacrylamide, 8 M urea, and 8868The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: Discussionmentioning

confidence: 99%

Dual regulation of open-complex formation and promoter clearance by Arc explains a novel repressor to activator switch.

Smith

Sauer

1996

Proc. Natl. Acad. Sci. U.S.A.

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“…As an activator, AraC in the presence of arabinose enhances expression. OmpR can activate or repress the ompF gene depending on osmolarity conditions and the strength of the promoter (30). Another protein, Lrp (the leucine-responsive regulatory protein), can be either an activator or repressor at Lrp target genes (21).…”

Section: Discussionmentioning

confidence: 99%

Identification of new genes regulated by the marRAB operon in Escherichia coli

Seoane

Levy

1995

J Bacteriol

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Random TnphoA and TnlacZ translational fusions were introduced into an Escherichia coli strain with a deletion of the multiple antibiotic resistance (mar) locus, complemented in trans by a temperature-sensitive plasmid bearing the mar locus with a constitutively expressed mar operon. Five gene fusions (two with lacZ and three with phoA) regulated by the mar operon were identified by increased or decreased marker enzyme activity following loss of the complementary plasmid at the restrictive temperature. Expression of LacZ from both lacZ fusions increased in the presence of the mar operon; expression from the three phoA fusions was repressed by the mar operon. The lacZ fusions were mapped at 31.5 and 14 min on the Escherichia coli chromosome. One of the phoA fusions was located at 51.6 min while the two others mapped at 77 min. Cloning and sequencing of a portion of the fused genes showed all of them to be different. Chromosomally mediated multiple antibiotic resistance (Mar) mutants of Escherichia coli constitutively express the regulated marRAB operon, which is located at 34 min on the E. coli chromosomal map (5, 13). Expression of the operon, consisting of marR, marA, and marB, is normally repressed by MarR but can be induced by diverse compounds such as tetracycline, chloramphenicol, menadione, and salicylates (6,16,28). Mar mutants show increased resistance to multiple antibiotics and structurally unrelated compounds (2,(12)(13)(14). Selected by low concentrations of a single drug, Mar mutants can attain very high levels of resistance if maintained in contact with the selective agent (12). Insertion of Tn5 into marA, the gene for a protein which causes increased antibiotic resistance (11,16,33), completely reversed the resistance phenotype (13).The marRAB operon affects distant chromosomal genes, as revealed by changes seen in proteins separated in two-dimensional gels (14). One effect is to increase expression of micF (8) and thereby indirectly decrease expression of ompF (8). However, the reduced level of OmpF in Mar mutants is only a small component of antibiotic resistance (6, 7), indicating that other genes are involved in the Mar phenotype. To identify the other genes regulated by the marRAB operon and involved in the Mar phenotype, we used a marker fusion approach, with TnphoA (22) and TnlacZ (32). We isolated fusions in which ␤-galactosidase or alkaline phosphatase expression changed when the mar operon was removed. As a result of these studies, we have identified four mar locus-regulated (mlr) genes, three of which have not previously been described. MATERIALS AND METHODSBacterial strains, plasmids, and phages. Bacterial strains and plasmids used in this study are listed in Table 1. The 1.24-kbp BspHI deletion in the mar locus in strain ASS110 was made by homologous recombination following methods described previously (17), with pWY4, a derivative of the temperature-sensitive plasmid pMAK705, bearing the deletion. The presence of the deletion in the recipient strain was verified by Southern blot anal...

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“…An observation consistent with our proposal was obtained with the OmpR transcriptional activator, which also stabilizes the binding of the RNA polymerase to the promoter as a closed complex. When the naturally activated weak promoter was artificially replaced by a strong promoter, the positive effect of OmpR turned to inhibitory (13) We are grateful to Dr. L. Rothman-Denes for critical reading ofthe manuscript, to Drs. M. Serrano and J. Ruiz for helpful discussions, to J. M. [izaro for purification ofRNA polymerase, and to L. Villar for technical assistance.…”

Section: Methodsmentioning

confidence: 99%

“…The transcription initiation process can be limited at different stages. Several transcriptional activators have been shown to act by favoring one or several of these rate-limiting steps (13)(14)(15)(16)(17)(18)(19). The molecular mechanisms leading to transcription activation are not fully understood.…”

Section: Rnpmentioning

confidence: 99%

Phage phi 29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein-protein contacts.

Nuez

Rojo

Salas

1992

Proc. Natl. Acad. Sci. U.S.A.

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Transcription from the late promoter, PA3, of Bacillus sublilis phage #29 is activated by the viral regulatory protein p4. A kinetic analysis of the activation process has revealed that the role of protein p4 is to stabilize the binding of RNA polymerase to the promoter as a cosed complex without significantly affecting further steps of the initiation process. Electrophoretic band-shift assays performed with a DNAfragment spanning only the protein p4 binding site showed that RNA polymerase could efficiently retard the complex formed by protein p4 bound to the DNA. Similarly, when a DNA fmraent containing only the RNA polymerase-binding region of PA3 was used, p4 greatly stimulated the binng of RNA polymerase to the DNA. These results strongly suggest that p4and RNA polymerase contact each other at the PA3 promoter.In the light of current knowledge of the p4 activation mechanism, we propose that direct contacts between the two proteins participate in the activation process. and the RNA polymerase have been proposed to participate in the activation process (3,6,18,(20)(21)(22)(23)(24)(25)(26)(27)(28). At the same time, DNA is thought to play an active role in the initiation mechanism by adopting three-dimensional structures that either directly accelerate one of the steps leading to transcription initiation or facilitate the correct stereospecific alignment of the activator and the RNA polymerase (4, [29][30][31][32][33] 11401The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

show abstract

Enhancement of RNA polymerase binding to promoters by a transcriptional activator, OmpR, in Escherichia coli: its positive and negative effects on transcription.

Cited by 29 publications

References 23 publications

Dual regulation of open-complex formation and promoter clearance by Arc explains a novel repressor to activator switch.

Dual regulation of open-complex formation and promoter clearance by Arc explains a novel repressor to activator switch.

Identification of new genes regulated by the marRAB operon in Escherichia coli

Phage phi 29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein-protein contacts.

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