The Purple Membrane Regulon in a Haloarchaeon

在这个项目中，遗传学和生物化学方法的结合被用于研究嗜盐古菌NRC-1中紫色膜的合成。紫色膜是细胞膜的一个特殊区域，它包含一种单色蛋白，细菌视紫红质（BR）的二维晶体阵列。BR由蛋白质片段（细菌视蛋白）和发色团（视网膜）的1:1复合物组成。当在微氧和高光照条件下完全诱导时，紫色膜可以覆盖细胞表面的50%，并能够支持一段时间的光养生长。此前，bop基因的转录依赖于上游基因bat，并发现对DNA超卷曲很敏感。此外，最小细菌视蛋白基因（bop）启动子被广泛诱变，并显示包含一个必需的上游激活子序列（UAS），一个偏离古细菌共识的TATA盒，以及一个位于嘌呤-嘧啶交替区域的DNA超卷曲敏感位点。可能的调节因子Bat含有PAS-PAC（或LOV）氧化还原传感结构域、GAF光响应结构域和AraC型螺旋-螺旋-螺旋（HTH） DNA结合基元的同源性。另外3个基因（crtB1、blp和brp）含有与bop基因相似的UAS元件，聚集在bop基因的上游，表现出类似或相同的调控特征，在基因组序列中发现6个tata结合蛋白（tbp）基因和7个TFIIB （tfb）基因。本项目的主要目标是在遗传水平上了解细菌视蛋白和视网膜的协调调节机制。具体而言，该项目将(1)确定与bop启动子结合的蛋白质，(2)确定多效性调节因子Bat与其他转录因子的作用机制，以及(3)确定紫色膜调节因子的成员。长期目标是更好地理解古细菌多基因系统的协调调节机制，这将有助于揭示所有生命分支的调节机制的进化。

0095700 DasSarma and SchreierIn 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.