Transcriptional factor Fur from Thermoplasma volcanium binds its own promoter DNA in a divalent cation-dependent manner

Abstract

Because archaea possess many respiratory enzymes or radical scavengers with catalytic domains that contain iron, the expression of the genes encoding these enzymes might be regulated by iron acquisition. The genome of an archaeon, Thermoplasma volcanium contains a gene that encodes Fur (TVN0292). The fur gene of T. volcanium was amplifi ed by PCR, and cloned into plasmid pET28a. TvFur (T. volcanium Fur protein) was expressed in E. coli cells and then purifi ed. EMSA revealed that TvFur binds to its own promoter DNA. The binding to its own promoter was in an Mn 2+ -, Zn

Specificity of Fur Binding to the Oxidative Stress Response Gene Promoter in the Facultative Anaerobic Archaeon <i>Thermoplasma volcanium</i>

“…These results indicate that the BRE and TA repeats in the TATA-box are essential for tvsod-ogg promoter recognition by TvFur. Together with the results of the previous study, 13) this suggests that TvFur recognizes these TA repeats.…”

“…1B). The dissociation constant for TvFur binding to its own promoter was approximately same value, 13) however that the constant for binding to the tvhemB promoter was 37% of this value (data not shown). These results indicate that TvFur binds to the former two promoters with higher affinities than to the tvhemB promoter region.…”

“…12) T. volcanium contains a gene encoding Fur (TVN0292, designated tvfur), and TvFur, the T. volcanium Fur protein, binds to its own promoter region in vitro. 13) In this study, we investigated the binding specificity of TvFur to the tvsod-ogg operon promoter by electrophoretic mobility shift assay (EMSA), and determined the TvFur binding sequence in this promoter by DNaseI footprinting analysis. TvFur recognized a TATA-box like TA repeat sequences.…”

“…The 5′ FAM labeled or non-labeled double-stranded DNA for EMSA was generated by annealing complementary oligonucleotides by incubation at 95°C for 5 min and then cooling to room temperature. EMSA was performed with 0.2 µM or 0.4 µM of DNA under essentially the same conditions as previously described 13) except electrophoresis buffer containing 1 mM dithiothreitol (DTT). The DNA bands were detected and analyzed with a Pharos FX molecular imager and Quantity One imaging software (Bio-Rad), and the dissociation constant (K d ) was calculated.…”

“…Determination of the DNA sequence of the protected region was performed according to a previously described method. 13) …”