Transcriptional Regulation of Carbon Utilization in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Blum，Paul MCB-9974453小亚基（16 S）核糖体RNA序列比较已经确定了一个独特的原核生物谱系或分组，称为微囊藻。 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远。 嗜热菌通常被细分为两个主要的类群，称为宽古菌和泉古菌;好氧超嗜热菌Sulfolobus solfataricus是泉古菌的一个被深入研究的成员。 超嗜热菌存在于两个原核生物群中，并以其根深蒂固（缓慢进化）的16 S rRNA区分。微生物与真核生物不同，但它们的一般（基础）转录组分包括RNA聚合酶、启动子和启动子结合蛋白之间存在显着的保守性。 这种进化重叠是否包括调节（辅助）转录成分尚不清楚。 本项目的研究重点是探讨酵母中基因表达的生物化学机制是否与真核生物或细菌中的机制相似，或者是采用全新的策略。太阳神 CR控制至少三种基因的表达，这些基因都编码用于在多糖上生长的葡糖基水解酶。 受影响的基因包括a-葡糖苷酶（malA）、a-淀粉酶（amyA）和b-糖苷酶（拉克）。 这些基因在转录水平上协同调节，以响应为生长提供的碳源的质量。 碳的质量是通过它被三氯乙酸循环吸收的容易程度来衡量的。随着碳源质量的降低，这些基因的表达增加（CR缓解）。由于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调节因子控制的。这种因子的存在进一步得到分离的非连锁（基因外）突变的支持，这些突变多效性地降低CR基因转录。 本项目将测试以下假设：1）CR通过顺式作用启动子序列的作用改变基因表达; 2）CR基因表达的变化需要反式作用因子的作用; 3）CR基因表达的差异调节是启动子特异性的。 实验方法将涉及CR依赖性和非依赖性基因之间的启动子区域的交换以及CR依赖性基因之间的启动子杂交体的构建。 将这些构建体与报道基因融合，并通过酶测定、蛋白质印迹和北方印迹进行体内分析。启动子将通过转录产物的引物延伸和凝胶阻滞试验进行体外分析。 预期的结果将提供深入了解古细菌超嗜热代谢和基因表达的进化起源的基本方面。

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.