PMID:7673113
| Citation |
Hidalgo, E, Bollinger, JM Jr, Bradley, TM, Walsh, CT and Demple, B (1995) Binuclear [2Fe-2S] clusters in the Escherichia coli SoxR protein and role of the metal centers in transcription. J. Biol. Chem. 270:20908-14 |
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| Abstract |
SoxR protein of Escherichia coli is activated by superoxide-generating agents or nitric oxide as a powerful transcription activator of the soxS gene, whose product activates approximately 10 other promoters. SoxR contains non-heme iron essential for abortive initiation of transcription in vitro. Here we show that this metal dependence extends to full-length transcription in vitro. In the presence of E. coli sigma 70 RNA polymerase, iron-containing SoxR mediates open complex formation at the soxS promoter, as determined using footprinting with Cu-5-phenyl-1,10-phenanthroline. We investigated the nature of the SoxR iron center by chemical analyses and electron paramagnetic resonance spectroscopy. Dithionite-reduced Fe-SoxR exhibited an almost axial paramagnetic signature with g values of 2.01 and 1.93 observable up to 100 K. These features, together with quantitation of spin, iron, and S2-, and hydrodynamic evidence that SoxR is a homodimer in solution, indicate that (SoxR)2 contains two [2Fe-2S] clusters. Treatment of Fe-SoxR with high concentrations of dithiothreitol caused subtle changes in the visible absorption spectrum and blocked transcriptional activity without generating reduced [2Fe-2S] centers, but was also associated with the loss of iron from the protein. However, lowering the thiol concentration by dilution allowed spontaneous regeneration of active Fe-SoxR. |
| Links | |
| Keywords |
Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Base Sequence; Copper/chemistry; DNA Primers; DNA, Superhelical; Electron Spin Resonance Spectroscopy; Escherichia coli/metabolism; Iron/metabolism; Iron-Sulfur Proteins/chemistry; Iron-Sulfur Proteins/metabolism; Molecular Sequence Data; Oxidative Stress; Phenanthrolines/chemistry; Sulfhydryl Compounds/chemistry; Templates, Genetic; Transcription Factors/chemistry; Transcription Factors/genetics; Transcription Factors/metabolism; Transcription, Genetic |
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