Gene Silencing and Catabolite Repression in the Archaeon Sulfolobus solfataricus

原核生物由细菌和古生菌组成。硫磺硫化叶菌（Sulfolobus solfataricus）是古细菌的泉古菌亚类的成员，其像真核生物一样使用Pol II型RNA聚合酶、TATA盒启动子和启动子结合蛋白催化基础转录。这种进化重叠是否包括调节（辅助）转录成分是未知的。 这个项目将集中在古生菌的基因调控机制及其与真核生物和细菌的关系。S. solfataricus将被用作古细菌基因调控的模型。CR系统对糖基水解酶（GH）基因的表达施加协调控制。改变生长激素的表达发生在响应碳源类型的变化（分解代谢物反应性），或通过在一个位点突变称为汽车。 car突变体表现出GH mRNA水平降低和分解代谢产物反应性减弱，表明car介导CR。由于GH mRNA的稳定性不受CR的影响，因此调控必须发生在转录合成水平。受影响基因的广泛基因组分布表明它们的协调表达采用了可能由car编码或调节的反式作用因子。 新的定向重组（基因置换）方法揭示了CR具有基因沉默的两个特征，即位置抑制和远距离作用。将受影响的GH基因重新定位到不同的染色体位点会破坏分解代谢产物的反应性，而插入到CAR调节位点的CAR不敏感启动子则会对CAR敏感（抑制）。本课题的主要假设是：S. Solfataricus利用真核转录沉默的祖先形式来控制基因表达。将使用基因置换和体外转录方法来验证这一假设，以鉴定和表征CR调节机制所使用的序列。该项目将通过扩大使用一个关于极端嗜热古菌的互联网网站，扩大代表性不足群体的参与，并扩大传播，以提高科学和技术认识。预期的项目结果将提供深入了解古菌代谢和基因表达的进化起源的基本方面。

Prokaryotes consist of bacteria and archaea. Sulfolobus solfataricus is a member of the crenarchaeal subdivision of the archaea which like eukaryotes catalyze basal transcription using a Pol II-type RNA polymerase, TATA-box promoters and promoter binding proteins. Whether this evolutionary overlap includes regulatory (accessory) transcription components is unknown. This project will focus on gene regulatory mechanisms in archaea and their relationship if any to eukaryotes and bacteria. The Catabolite Repression (CR) system of S. solfataricus will be used as a model of archaeal gene regulation. The CR system exerts coordinate control over the expression of glycosyl hydrolase (GH) genes. Altered GH expression occurs in response to changes in the type of carbon source (catabolite responsiveness), or by mutation at a locus called car. car mutants exhibit reduced GH mRNA levels and an attenuation of catabolite responsiveness suggesting car mediates CR. Since GH mRNA stability is unaffected by CR, regulation must occur at the level of transcript synthesis. Widespread genomic distribution of the affected genes indicates their coordinate expression employs a trans-acting factor possibly encoded or regulated by car. New directed recombination (gene replacement) methods reveal CR exhibits two characteristics of gene silencing, positional repression and action-at-a-distance. Relocation of an affected GH gene to a different chromosomal locus disrupts catabolite responsiveness, while a car-insensitive promoter inserted at a car-regulated locus becomes sensitive (repressed) by car. The main hypothesis of this project is that CR in S. solfataricus employs an ancestral version of eukaryotic transcriptional silencing to control gene expression. This hypothesis will be tested using gene replacement and in vitro transcription methods to identify and characterize sequences used by the CR regulatory mechanism. The project will broaden inclusion of under represented groups and broaden dissemination to enhance scientific and technological understanding by expanding use of a web-based internet site on hyperthermophilic archaea. The anticipated project results will provide insight into basic aspects of archaeal metabolism and the evolutionary origin of gene expression.