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Cloning and characterization of nickel uptake regulator NUR mutants from Streptomyces coelicolor
| Content Provider | Semantic Scholar |
|---|---|
| Author | Manley, Olivia M. Grossoehme, Nick |
| Copyright Year | 2015 |
| Abstract | Sufficient concentrations of metal within a cell are required for proper cellular function; however, metals become toxic at very high concentrations. Therefore, it is important for an organism to have a mechanism for maintaining metal homeostasis within its cells. Streptomyces coelicolor, a soil-dwelling bacterium important in the production of antibiotics, utilizes the nickel uptake regulator (NUR) to maintain nickel homeostasis and oxidative response. NUR functions as a transcriptional repressor that responds to changing cytosolic concentrations of Ni. Previous research describes two key metal-binding sites per NUR monomer. Our research seeks to analyze how changes to these binding sites affect the ability of NUR to coordinate metals and bind to DNA. NUR mutants containing single or multiple amino acid substitutions at each binding site were cloned. Biophysical characterization of these mutants and the wild-type protein will aid in understanding the role each metal site plays in the function of NUR. INTRODUCTION Biological Significance of Metals Various metal ions are essential nutrients to cells, such as Fe, Zn, Mn, Cu, Co, Mo, and Ni, while other metals are toxic to a cell, such as Cd, Hg, Ag, Pb, Sn, and Cr. All metals are necessary for the cell, but become toxic at too high of a concentration, importantly Zn, Cu, and Fe. Metal toxicity often results from toxic metals displacing nutrient metals from their metabolic site as the concentration of toxic metals grows.1 Many metals aid in the catalysis of various biological reactions necessary for cellular function, but the concentration of these metals must be tightly regulated to maintain homeostasis.2 High concentrations of nickel, for example, become carcinogenic as a result of the formation of superoxide species and may damage DNA or oxidize lipids.3 As a result of the dual nature of metals as both essential and toxic, it is highly important to regulate the levels of nickel, as well as other metals, within the cell. Transcriptional Regulators Regulation of metal concentrations within the cell begins at the level of transcription. Transcription factors are molecules, typically proteins, that control whether or not transcription occurs. These regulators often consist of a site that binds to DNA and a site that binds to a cofactor. The binding of the cofactor is related to the levels of stimuli and affects the ability of the protein to bind to DNA, thus regulating the response to these stimuli.4 For example, as the concentration of zinc becomes too great, zinc ions will bind to a transcription factor that blocks the transcription of zinc-uptake proteins.5 The portion of the DNA that the transcription factor binds to is called the promoter. The promoter contains a specific base pair sequence recognized by the transcription factor and the transcription initiation site that indicates where transcription of the DNA should begin.6 Transcription factors can either be activators or repressors. Activators promote transcription, while repressors prevent transcription. RNA polymerase is the enzyme that synthesizes mRNA from DNA to later direct the construction of proteins.4 An activator directs RNA polymerase to begin transcription of a specific gene or quickens the rate at which RNA polymerase functions. A repressor prevents transcription, binding to activators to negate their effect or competing with activators for target DNA.6 Metalloregulators are a critical type of transcription factor in regulating intracellular levels of metals. Metalloregulators serve an important role in bacteria, as bacteria may experience drastic changes |
| Starting Page | 9 |
| Ending Page | 9 |
| Page Count | 1 |
| File Format | PDF HTM / HTML |
| Volume Number | 1 |
| Alternate Webpage(s) | https://digitalcommons.winthrop.edu/cgi/viewcontent.cgi?article=1006&context=wmrb&httpsredir=1&referer= |
| Alternate Webpage(s) | https://digitalcommons.winthrop.edu/cgi/viewcontent.cgi?article=1006&context=wmrb |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
