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Abstract
The archaeal basal transcription apparatus is eukaryotic in nature while transcriptional regulation appears to occur primarily through bacteria-like mechanisms; however, only a small number of archaeal transcriptional regulators have been characterized in detail. This work describes the discovery and characterization of an additional archaeal transcriptional regulator, SurR, of the hyperthermophile Pyrococcus furiosus. Much of the gene regulation which occurs at the transcriptional level is the result of regulatory proteins; therefore, a DNA affinity protein capture experiment was employed to capture transcription factors responsible for the observed gene regulation in DNA microarray expression profiles of the transcriptional response of P. 0furiosus to growth in the presence or absence of elemental sulfur (S). The upstream DNA from 0the first ORF in the S down-regulated membrane-bound hydrogenase operon (mbh1) was used as bait to capture and identify SurR from P. furiosus cell extracts derived from cultures grown 0in the presence and absence of S. The recombinant protein was verified to bind specifically to the mbh1 promoter by electromobility shift assay and fluorescence-detected DNase I footprinting. Elucidation of its recognized DNA-binding motif by SELEX allowed the detection and verification of other binding sites in the genome, many of which were found upstream from 0the ORFs which are up- and down-regulated within 10 min of S addition to a growing P. furiosus culture as determined by DNA microarray. In vitro transcription with two of these ORFs demonstrated that SurR is both a transcriptional activator and repressor. The X-ray crystal structure revealed the presence of a disulfide bond at the CxxC motif in the N-terminal DNA binding domain. This motif was found to act as a redox switch sensitive to colloidal sulfur, such that oxidation of SurR abolishes its sequence specific DNA binding affinity. The work presented here demonstrates that this novel transcriptional regulator, termed SurR for Sulfur-response regulator, is likely a relevant participant in transcriptional regulation pathways related to P. 0furiosus S metabolism.