Gene Silencing and Catabolite Repression in the Archaeon Sulfolobus solfataricus

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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.