Reconstitution of Pyrococcus furiosus RNA polymerase and identification of potential transcriptional regulators responding to iron deprivation in Pyrococcus furiosus

As part of a large-scale effort to discover regulatory mechanisms in the hyperthermophilic archaeon Pyrococcus furiosus (Pf), (1) Pf RNA polymerase (RNAP) subunits (10) were cloned, expressed, purified and reconstituted to form Pf RNAP complex; (2) DNA-affinity protein capture method was used to identify PF0851p as a potential regulatory transcription factor associated with PF1281, PF1282, PF1283 operon (PF1282, encoded as rubredoxin, down-regulated under iron limitation based on Microarray expression profiling) and PF1287, PF1285, PF1286 operon (PF1285, PF1286, putative sufD, sufB respectively, up-regulated under iron limitation based on Microarray expression profiling). The binding potential of PF0851p on PF1283 UOR and PF1287 UOR has been determined by electromobility shift assay and the putative binding site for both the UORs were determined by DNase I footprinting assay.