Gene Silencing and Catabolite Repression in the Archaeon Sulfolobus solfataricus

原核生物由细菌和古菌组成。Sulfolobus solfararicus是古生菌中的一员，它与真核生物一样，利用POLII型RNA聚合酶、TATA-box启动子和启动子结合蛋白来催化碱基转录。这种进化重叠是否包括调控(辅助)转录成分尚不清楚。该项目将重点研究古细菌中的基因调控机制，以及它们与真核生物和细菌的关系。解冻链球菌的分解代谢抑制(CR)系统将被用作古生菌基因调控的模型。CR系统对糖基水解酶(GH)基因的表达进行协调控制。生长激素表达的改变是对碳源类型(分解代谢反应)的反应，或者是通过一个名为CAR的基因突变来发生的。CAR突变体表现出生长激素mRNA水平的降低和分解代谢反应性的减弱，表明CAR介导了CR。由于生长激素mRNA的稳定性不受CR的影响，因此必须在转录合成水平上进行调节。广泛分布的受影响基因的基因组分布表明，它们的协调表达使用了一个反式作用因子，可能由CAR编码或调节。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。将受影响的生长激素基因重新定位到不同的染色体位置会破坏分解代谢的反应，而插入在CAR调节的基因座上的CAR不敏感的启动子则会被CAR敏感(抑制)。这个项目的主要假设是，索尔法特链霉菌中的CR利用真核转录沉默的祖先版本来控制基因的表达。这一假说将使用基因替换和体外转录方法进行验证，以识别和表征CR调节机制使用的序列。该项目将扩大对代表性不足群体的纳入，并扩大传播，通过扩大使用一个关于高温古生菌的因特网网站来加强对科学技术的了解。预期的项目结果将提供对古生物新陈代谢的基本方面和基因表达的进化起源的洞察。

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.