A Design for Life: Prokaryotic Metal-binding MerR Family Regulators

Hobman, Jon L.; Wilkie, John; Brown, Nigel L.

doi:10.1007/s10534-005-3717-7

Cited by 94 publications

(107 citation statements)

References 46 publications

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“…It is worth noting that cellular conditions are not exactly the same as in our experiments and kinetic constants may thus differ; nevertheless, our results show that both the direct protein substitution and the assisted protein dissociation are possible pathways for turning off transcription. Past studies have shown that for the prototype Hg 2þ -responsive metalloregulator MerR, a protein called MerD might mediate the unbinding of holo-MerR from DNA for turning off transcription (28). No evidence has yet been found, however, for a MerD homologue for CueR or other MerR-family regulators.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Metals play a critical role in bacterial life as essential micronutrients fundamental to central metabolic processes and are required for pathogenic virulence (1,41,45). Intricate machinery has evolved to finely tune the intracellular concentration of metals to achieve metal homeostasis and prevent metal toxicity (35).…”

Section: Rna Polymerase Is Not Poised At Lag-inducible Promotersmentioning

confidence: 99%

Lag Phase Is a Distinct Growth Phase That Prepares Bacteria for Exponential Growth and Involves Transient Metal Accumulation

et al. 2012

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Lag phase represents the earliest and most poorly understood stage of the bacterial growth cycle. We developed a reproducible experimental system and conducted functional genomic and physiological analyses of a 2-h lag phase in Salmonella enterica serovar Typhimurium. Adaptation began within 4 min of inoculation into fresh LB medium with the transient expression of genes involved in phosphate uptake. The main lag-phase transcriptional program initiated at 20 min with the upregulation of 945 genes encoding processes such as transcription, translation, iron-sulfur protein assembly, nucleotide metabolism, LPS biosynthesis, and aerobic respiration. ChIP-chip revealed that RNA polymerase was not "poised" upstream of the bacterial genes that are rapidly induced at the beginning of lag phase, suggesting a mechanism that involves de novo partitioning of RNA polymerase to transcribe 522 bacterial genes within 4 min of leaving stationary phase. We used inductively coupled plasma mass spectrometry (ICP-MS) to discover that iron, calcium, and manganese are accumulated by S. Typhimurium during lag phase, while levels of cobalt, nickel, and sodium showed distinct growth-phase-specific patterns. The high concentration of iron during lag phase was associated with transient sensitivity to oxidative stress. The study of lag phase promises to identify the physiological and regulatory processes responsible for adaptation to new environments.

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“…Many others are required for cellular function, such as Zn and Cu, but can be toxic in excess. Cells have thus developed many ways to regulate intracellular metal concentrations (1)(2)(3)(4)(5)(6)(7)(8)(9). Metal-responsive transcriptional regulation is one of them, where metalloregulators respond to intracellular metal ions and regulate transcription of metal efflux, uptake, or other metal homeostasis genes (4)(5)(6)(7)(8)(9).…”

mentioning

confidence: 99%

Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription

Martell

Joshi

Gaballa

et al. 2015

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

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Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ 70 -dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator−DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)-a Cu + -responsive MerR-family metalloregulator-modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR−PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the deadend and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo-and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.single-molecule FRET | protein-DNA interaction dynamics | MerR-family regulators | metal-responsive transcription regulation M aintaining cellular metal homeostasis is essential for bacteria, which often dwell in environments with high concentrations of metals. Some of these metals are purely toxic to bacteria, such as Cd and Hg. Many others are required for cellular function, such as Zn and Cu, but can be toxic in excess. Cells have thus developed many ways to regulate intracellular metal concentrations (1-9). Metal-responsive transcriptional regulation is one of them, where metalloregulators respond to intracellular metal ions and regulate transcription of metal efflux, uptake, or other metal homeostasis genes (4-9).In Gram-negative bacteria, MerR-family metalloregulators act on σ 70 -dependent suboptimal promoters to repress or activate transcription of metal resistance genes (7,8). These suboptimal promoters have elongated spacing, 19-20 bp (Fig. 1), compared with the optimal 17 ± 1 bp, between the −35 and −10 elements. This elongated spacing causes a misalignment of these two recognition elements, impairing proper interactions with the RNA polymerase (RNAP) and leading to a weak basal leve...

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A Design for Life: Prokaryotic Metal-binding MerR Family Regulators

Cited by 94 publications

References 46 publications

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

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

Lag Phase Is a Distinct Growth Phase That Prepares Bacteria for Exponential Growth and Involves Transient Metal Accumulation

Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription

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