Identification and characterization of a new transcription factor involved in Pyrococcus furiosus response to oxidative stress
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Gene expression is often under regulation at the transcriptional level, controlled by transcription factors. The primary goal of this research project is to discover and characterize new transcription factors in Pyrococcus furiosus (Pf), a model archaeal organism. The transcription system of archaea has both bacterial-like and eukaryotic-like features. Only a very limited number of archaeal transcription regulatory proteins have been identified to date, and transcription regulatory pathways are far from being well known. In this project, transcription factors were discovered using DNA affinity protein capture followed by mass spectrometry based protein identification. The Pf gene expression profile on the genome-wide scale has been described and compared between two different cell-growth conditions with cumene hydroperoxide and without cumene hydroperoxide. Additional bioinformatics analysis identified a conserved palindromic sequence in the putative promoter region of several genes that are upregulated by growth with cumene hydroperoxide and all are involved in oxidative stress. Based on these analyses, the DNA fragments upstream of the open reading frame of PF1983 and PF1513, which both encode hypothetical protein, were selected for DNA-affinity protein capture. A novel transcription regulatory protein PF0230p was identified and its specific DNA binding ability at sites upstream of multiple genes was confirmed by the electrophoretic mobility shift assay (EMSA). DNase I footprinting showed that PF0230p binds to a region upstream of both PF1983 and PF1513, which contains the conserved palindromic motif, ATTAAT. Furthermore, PF0230p was shown to repress PF1983 but enhance PF1513 gene transcription in cell free transcription assays. These results suggest PF0230p is a transcriptional regulator recognizing a conserved palindromic motif and possibly regulating a group of genes in the oxidative stress pathway in Pyrococcus furiosus.