0095700 DasSarma and Schreier In this project, a combination of genetic and biochemical approaches are being used to investigate purple membrane synthesis in a salt-loving archaeon, Halobacterium NRC-1. The purple membrane is a specialized region of the cell membrane that contains a two-dimensional crystalline array of a single chromoprotein, bacteriorhodopsin (BR). BR is composed of a 1:1 complex of the protein moiety (bacterio-opsin) and chromophore (retinal). When fully induced under microaerobic and high illumination conditions, purple membrane can cover up to 50 % of the cell's surface and is able to support a period of phototrophic growth. Previously, transcription of the bop gene was shown to depend upon an upstream gene, bat, and found to be sensitive to DNA supercoiling. Additionally, the minimal bacterio-opsin gene (bop) promoter was extensively mutagenized and shown to contain an essential upstream activator sequence (UAS), a TATA box deviating from the archaeal consensus, and a DNA supercoiling-sensitivity site within an alternating purine-pyrimidine region. The likely regulator, Bat, contains homologies to the PAS-PAC (or LOV) redox sensing domain, a GAF light-responsive domain, and an AraC type helix-turn-helix (HTH) DNA binding motif. Three other genes (crtB1, blp, and brp) containing UAS elements similar to the bop gene, and clustered immediately upstream of bop, showed similar or identical regulatory characteristics, and six TATA-binding protein (tbp) genes and seven TFIIB (tfb) genes were found in the genome sequence. The major goal of this project is to understand the mechanism of coordinate regulation of bacterio-opsin and retinal at the genetic level. Specifically, the project will (1) identify the proteins binding to the bop promoter, (2) define the mechanism of action of the pleiotropic regulator, Bat, in concert with other transcriptional factors, and (3) establish the members of the purple membrane regulon. The long-term goal is to better understand the mechanisms of coordinate regulation of multigene systems in archaea, which should shed light on the evolution of regulatory mechanisms across all branches of life.
