Transcriptional Regulation of Carbon Utilization in the Hyperthermophilic Archaeon Sulfolobus solfataricus

小亚基(16S)核糖体RNA序列比较已经确定了一种独特的原核生物谱系或群体，称为古生菌。它们与细菌的亲缘关系就像它们与真核生物的亲缘关系一样遥远。古生菌通常被分为两大类群，即真古生菌和新古生菌；好氧嗜热菌Sulfolobus solfararicus是新古生菌中研究得最深入的成员。嗜热菌存在于这两个原核生物组中，其特点是根深蒂固(缓慢进化)的16S rRNA。古生菌不同于真核生物，但它们的一般(基本)转录成分之间有显著的保守性，包括RNA聚合酶、启动子和启动子结合蛋白。这种进化重叠是否包括调控(辅助)转录成分尚不清楚。本项目的重点是研究古生菌中基因表达的生化机制是否与真核生物或细菌中的机制类似，或者是采用全新的策略。在先前的研究中，在S.solfararicus中发现了一个分解代谢抑制(CR)基因调控系统。CR控制至少三个基因的表达，这三个基因都编码葡萄糖水解酶，用于生长在多糖上。受影响的基因包括a-葡萄糖苷酶(MalA)、a-淀粉酶(AMYA)和b-糖苷酶(Lacs)。这些基因在转录水平上受到协调调节，以响应为生长提供的碳源的质量。碳质量的衡量标准是它被吸收到三氯乙烷循环中的难易程度。随着碳源质量的降低，这些基因的表达增加(CR得到缓解)。因为这些基因在物理上是不相连的，协调表达表明它们是由反式作用的调节因子控制的。分离的非连锁(基因外)突变多向性地减少CR基因转录，进一步支持了这种因子的存在。本项目将检验以下假设：1)CR通过作用于顺式作用的启动子序列改变基因的表达；2)CR基因表达的改变需要反式作用因子的作用；以及3)CR基因表达的差异调控是启动子特有的。该实验方法将需要CR依赖和独立基因之间的启动子区域的交换，以及CR依赖基因之间的启动子杂交的构建。这些构建物将被融合到一个报告基因，并在体内通过酶分析、Western印迹和Northern印迹进行分析。启动子将通过转录产物的引物延伸和凝胶滞留试验进行体外分析。预期的结果将提供对古生菌高温代谢的基本方面的洞察和基因表达的进化起源。

Blum, Paul MCB-9974453Small subunit (16S) ribosomal RNA sequence comparisons have identified a unique lineage or grouping of prokaryotic organisms called Archaea. They are as distantly related to Bacteria as they are to Eukaryotes. Archaea generally are subdivided into two major groups called the Euryarchaea and the Crenarchaea; the aerobic hyperthermophile, Sulfolobus solfataricus, is an intensively studied member of the Crenarchaea. Hyperthermophiles are present in both prokaryotic groups and are distinguished by their deeply rooted (slowly evolving) 16S rRNA. Archaea are distinct from Eukaryotes yet there is remarkable conservation between their general (basal) transcription components including RNA polymerase, promoters and promoter binding proteins. Whether this evolutionary overlap includes regulatory (accessory) transcription components is not known. This project focuses on the question of whether the biochemistry of gene expression in Archaea employs mechanisms like those in Eukaryotes or in Bacteria, or instead, altogether new strategies.In previous studies a Catabolite Repression (CR) gene regulatory system was discovered in S. solfataricus. CR controls expression of at least three genes all encoding glucosyl hydrolases which are used for growth on polysaccharides. The affected genes include an a-glucosidase (malA), an a-amylase (amyA), and a b-glycosidase (lacS). These genes are coordinately regulated at the transcriptional level in response to the quality of the carbon source provided for growth. Carbon quality is measured by how readily it can be assimilated into the TCA cycle. As carbon source quality decreases, expression of these genes is increased (CR is relieved). Because these genes are physically unlinked, coordinated expression indicates they are controlled by a trans-acting regulatory factor. The existence of such a factor is further supported by the isolation of unlinked (extragenic) mutations which pleiotropically reduce CR gene transcription. This project will test the following hypotheses: 1) CR alters gene expression through action at cis-acting promoter sequences; 2) Changes in CR gene expression require the action of a trans-acting factor; and 3) Differential regulation of CR-gene expression is promoter specific. The experimental approach will entail the exchange of promoter regions between CR-dependent and independent genes and the construction of promoter hybrids between CR-dependent genes. These constructs will be fused to a reporter gene and analyzed in vivo by enzyme assays, western blot and northern blot. Promoters will be analyzed in vitro by primer extension of transcription products and by gel retardation assays. The anticipated results will provide insight into basic aspects of archaeal hyperthermophilic metabolism and the evolutionary origin of gene expression.