Position and density effects on repression by stationary and mobile DNA-binding proteins.

Elledge, Stephen J.; Davis, Ronald W.

doi:10.1101/gad.3.2.185

Cited by 109 publications

(103 citation statements)

References 51 publications

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“…A variation of the transcriptional interference assay of Elledge and Davis (39), where repression of an antisense transcript by a DNAbinding protein leads to the expression of spectinomycin resistance, was used to assess in vivo binding. This assay has the advantage of probing for DNA binding under physiological conditions, but comparing the results with the in vitro data indicates that binding to the antisense promoter may not be sufficient to give spectinomycin resistance.…”

Section: Discussionmentioning

confidence: 99%

“…The single copy reporter plasmid (pPVUII388) compatible with the PvuII expression vector was a derivative of pNN388 (39). This plasmid carried one copy of the aadA gene (spectinomycin resistance) with a strong antisense promoter containing a unique PvuII recognition site as an operator.…”

Section: Methodsmentioning

confidence: 99%

“…Binding of Mutants to the PvuII Site-DNA binding was tested with a variation of the transcriptional interference assay (26,39); binding of a catalytically deficient mutant to a PvuII site on a reporter plasmid allowed the expression of spectinomycin resistance (see "Experimental Procedures"). Two mutants gave a significant indication of in vivo binding: D34G and H85A (Table I).…”

Section: Analysis Of Interaction Between Pvuii and Its Specific Bindimentioning

confidence: 99%

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Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Nastri

Evans

Walker

et al. 1997

Journal of Biological Chemistry

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The role of particular residues of the PvuII endonuclease in DNA binding and cleavage was studied by mutational analysis using a number of in vivo and in vitro approaches. While confirming the importance of residues predicted to be involved directly in function by the crystal structure, the analysis led to several striking results. Aspartate 34, which contacts the central base pair of the PvuII site (5 -CAGCTG-3 ) through the minor groove, plays a critical role in binding specificity. A D34G mutant binds with high affinity to any of the sequences in the set CANNTG, although its low level of cleavage activity acts only on the wild-type site. In addition, a His to Ala mutation at the residue that contacts the central G and is predicted to be blocked by PvuII methylation still requires the PvuII methylase to be maintained in vivo, arguing against this hypothesis as the only mechanism for methylation protection. Finally, four of the five mutations that reduce cleavage activity while still exhibiting binding in the gel shift assay are at residues that form DNA-or subunit-subunit contacts rather than in the catalytic center. This provides further evidence for a strong linkage between specific binding and catalysis.The structures of the five type II endonucleases determined by x-ray diffraction, EcoRI (1), BamHI (2, 3), EcoRV (4, 5), PvuII (6, 7) and Cfr10I (8), share substantial elements of similarity. Analysis of the enzymes complexed to DNA, where known, suggests a preliminary classification into two different groups (9). The endonucleases that produce 5Ј-overhanging ends, EcoRI and BamHI, approach the DNA from the major groove, where most of their base-specific contacts are made. The endonucleases that produce blunt-ended DNA products, EcoRV and PvuII, approach the DNA from the minor groove side and establish contacts in the major groove by wrapping around the DNA. Structure-function studies have been limited to a small number of type II enzymes. Years of studies have produced an extensive amount of information about EcoRI (10 -18), EcoRV (18 -24), and NaeI (25) and, more recently, BamHI (26,27). These studies have focused on the identification and analysis of residues involved in catalysis, testing alternative mechanisms of catalysis, and understanding how a specific DNA sequence is recognized. Attempts to alter the sequence specificity have proven to be difficult, with successful examples limited to mutants showing relaxed specificity (10,11,28) or displaying activity toward DNA substituted with unnatural bases (15,29). One explanation for this difficulty is that a change in specificity not only requires recognition of a different DNA sequence, but also requires that the linkage between recognition and catalysis be retained. A class of mutations that might illuminate this linkage is the catalytic mutations, where specific DNA binding is retained but catalysis is reduced (26, 30). In particular, mutants in this class that are outside the catalytic center might be deficient in the linkage between recognition and ca...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Analysis Of Interaction Between Pvuii and Its Specific Bindimentioning

confidence: 99%

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Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Nastri

Evans

Walker

et al. 1997

Journal of Biological Chemistry

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“…The ramA promoter, including the total ramR sequence, and the soxS promoter were amplified by PCR from the genomic DNA of strain ATCC 14028s using LA-Tag Polymerase (Takara Bio) and primers soxS-F-NotI, soxS-R-HindIII, ramA-F-NotI and ramA-R-HindIII (Table 2). The PCR fragment was cloned between the NotI and HindIII sites of the pNN387 vector (Elledge & Davis, 1989). The two mutations, RamRC67S and RamRC134S, were obtained by an inverse PCR-based site-directed mutagenesis using the KOD-Plus-Mutagenesis kit (Toyobo) and primers ramR-C67S-F, ramR-C67S-R, ramR-C134S-F and ramR-C134S-R (Table 2).…”

Section: Methodsmentioning

confidence: 99%

Regulation of the AcrAB multidrug efflux pump in Salmonella enterica serovar Typhimurium in response to indole and paraquat

et al. 2011

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Salmonella enterica serovar Typhimurium has at least nine multidrug efflux pumps. Among these, AcrAB is constitutively expressed and is the most efficient, playing a role in both drug resistance and virulence. The acrAB locus is induced by indole, Escherichia coli-conditioned medium, and bile salts. This induction is dependent on RamA through the binding sequence in the upstream region of acrA that binds RamA. In the present study, we made a detailed investigation of the ramA and acrAB induction mechanisms in Salmonella in response to indole, a biological oxidant for bacteria. We found that acrAB and ramA induction in response to indole is dependent on RamR. However, the cysteine residues of RamR do not play a role in the induction of ramA in response to indole, and the oxidative effect of indole is therefore not related to ramA induction via RamR. Furthermore, we showed that paraquat, a superoxide generator, induces acrAB but not ramA. We further discovered that the mechanism of acrAB induction in response to paraquat is dependent on SoxS. The data indicate that there are at least two independent induction pathways for acrAB in response to extracellular signals such as indole and paraquat. We propose that Salmonella utilizes these regulators for acrAB induction in response to extracellular signals in order to adapt itself to environmental conditions.

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“…After digestion with NotI and HindIII (Promega), the gel-purified fragment was ligated into the plasmid pNN387 (12), which was treated with the same restriction enzymes such that the expression of the lacZ gene on the plasmid is driven by the tna promoter. Positive clones were screened by colony PCR and verified by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

The Multidrug Efflux Pump MdtEF Protects against Nitrosative Damage during the Anaerobic Respiration in Escherichia coli

Zhang

Xiao

Horiyama

et al. 2011

Journal of Biological Chemistry

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Drug efflux represents an important protection mechanism in bacteria to withstand antibiotics and environmental toxic substances. Efflux genes constitute 6 -18% of all transporters in bacterial genomes, yet the expression and functions of only a handful of them have been studied. Among the 20 efflux genes encoded in the Escherichia coli K-12 genome, only the AcrABTolC system is constitutively expressed. The expression, activities, and physiological functions of the remaining efflux genes are poorly understood. In this study we identified a dramatic up-regulation of an additional efflux pump, MdtEF, under the anaerobic growth condition of E. coli, which is independent of antibiotic exposure. We found that expression of MdtEF is upregulated more than 20-fold under anaerobic conditions by the global transcription factor ArcA, resulting in increased efflux activity and enhanced drug tolerance in anaerobically grown E. coli. Cells lacking mdtEF display a significantly decreased survival rate under the condition of anaerobic respiration of nitrate. Deletion of the genes responsible for the biosynthesis of indole, tnaAB, or replacing nitrate with fumarate as the terminal electron acceptor during the anaerobic respiration restores the decreased survival of ⌬mdtEF cells. Moreover, ⌬mdtEF cells are susceptible to indole nitrosative derivatives, a class of toxic byproducts formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions. Taken together, we conclude that the multidrug efflux pump MdtEF is up-regulated during the anaerobic physiology of E. coli to protect the bacterium from nitrosative damage through expelling the nitrosyl indole derivatives out of the cells.

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Position and density effects on repression by stationary and mobile DNA-binding proteins.

Cited by 109 publications

References 51 publications

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Catalytic and DNA Binding Properties of PvuII Restriction Endonuclease Mutants

Regulation of the AcrAB multidrug efflux pump in Salmonella enterica serovar Typhimurium in response to indole and paraquat

The Multidrug Efflux Pump MdtEF Protects against Nitrosative Damage during the Anaerobic Respiration in Escherichia coli

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