DNA Distortion Mechanism for Transcriptional Activation by ZntR, a Zn(II)-responsive MerR Homologue in Escherichia coli

Outten, Caryn E.; Outten, F. Wayne; O’Halloran, Thomas V.

doi:10.1074/jbc.274.53.37517

Cited by 185 publications

(206 citation statements)

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“…2b, c). Both the extension of the protected region (from nt 239 to 213 relative to the transcription start site in the coding strand and from nt 215 to 240 in the noncoding strand) and the presence of hypersensitive bands (at nt 231 and 219 and at 233 and 221 in the coding and non-coding strands, respectively) are common features of the protein-DNA interaction described for the MerR family (Ansari et al, 1995;O'Halloran et al, 1989;Outten et al, 1999). The protected sequence of the copA promoter encompasses the sequence 59-TTGACCTTA-ACCTTGCTGGAAGGTTTA-39, which includes an imperfect ACCTTCC inverted repeat sequence located between the predicted 235 and 210 elements in the copA promoter region, is similar to the predicted E. coli CueR-binding site ( Fig.…”

Section: Expression Of Copa In Salmonella Is Directly Controlled By Cuermentioning

confidence: 99%

Dissecting the Salmonella response to copper

et al. 2007

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Section: Expression Of Copa In Salmonella Is Directly Controlled By Cuermentioning

confidence: 99%

Dissecting the Salmonella response to copper

et al. 2007

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“…Bacteria achieve the delicate balance between the requirement for Zn and its toxicity by the coordinated action of high-and low-affinity uptake systems and of export systems that rid the cells of excess Zn. Thus, different transporters acquire the metal from the growth medium to reach a total concentration in the submillimolar range, while transcriptionally controlled zinc uptake and efflux systems maintain the readily exchangeable "free" zinc at very low concentrations [3]. Whereas initial in vitro studies suggested that cellular "free" zinc levels are in the femtomolar range [3], more recent studies involving ratiometric zinc biosensors have shown that the in vivo "free" zinc is around 20 pM [4].…”

Section: Introductionmentioning

confidence: 99%

The Salmonella enterica ZinT structure, zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc

Ilari

Alaleona

Tria

et al. 2014

Biochimica et Biophysica Acta (BBA) - General Subjects

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This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. Background: In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT-ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim. Methods: ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy). Results: The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a K D value of 22 ± 2 nM for ZinT, while those with ZnuA point to one high affinity (K D b 20 nM) and one low affinity Zn(II) binding site (K D in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT-ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other. Conclusions: ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA. General significance: Knowledge of the ZinT-ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.

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“…They regulate transcription via a DNA distortion mechanism (5,(13)(14)(15)(16). They recognize specific dyad-symmetric DNA sequences within a promoter, and both their apo and holo forms bind DNA tightly.…”

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confidence: 99%

Direct substitution and assisted dissociation pathways for turning off transcription by a MerR-family metalloregulator

Joshi¹,

Panda²,

Martell³

et al. 2012

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

107

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Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu þ -responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.single-molecule imaging | protein-DNA interaction dynamics B acteria often dwell in environments with high concentrations of metals. Some of these metals are essential, but many are toxic. Even the essential metals, for example iron and copper, can become detrimental above a certain concentration inside cells. Many biological processes are thus present to regulate and maintain intracellular metal homeostasis (1-9). One of them is through metalloregulators, which respond to metal ions and regulate the transcription of genes that protect the bacteria from metal-induced stress (5-7, 10). The MerR-family metalloregulators respond to many metal ions with high selectivity and sensitivity, such as Hg 2þ and Cu 2þ (5,11,12).All MerR-family metalloregulators are homodimeric proteins. They regulate transcription via a DNA distortion mechanism (5, 13-16). They recognize specific dyad-symmetric DNA sequences within a promoter, and both their apo and holo forms bind DNA tightly. In the absence of metal, the metalloregulator bends the DNA; in this configuration RNA polymerase (RNAp) cannot interact with both −10 and −35 sequences properly and transcription is repressed. Upon binding metal, the metalloregulator changes its conformation and further unwinds the DNA slightly to allow proper RNAp interactions with the −10 and −35 sequences; transcription is then activated.Although the mechanisms of transcription activation by MerRfamily metalloregulators are well-studied (5, 13-16), little is yet known about how transcription activation is reversed. Turning off transcription promptly is important, however, as the cells would not want to conti...

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DNA Distortion Mechanism for Transcriptional Activation by ZntR, a Zn(II)-responsive MerR Homologue in Escherichia coli

Cited by 185 publications

References 60 publications

Dissecting the Salmonella response to copper

Dissecting the Salmonella response to copper

The Salmonella enterica ZinT structure, zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc

Direct substitution and assisted dissociation pathways for turning off transcription by a MerR-family metalloregulator

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