The archaea, or archaebacteria, were recognized as a form of life distinct from bacteria and eucarya just two decades ago. Studies of the archaeal transcription apparatus indicate that the archaeal system is highly homologous to the system in eucarya. However, little is known about the detailed mechanism of transcription or its regulation in the archaea. The experiments in this project attempt to address these questions by defining the topographical structure of the transcription complex from the hyperthermophilic archaeon Pyrococcus furiosus. The arrangement of the RNA polymerase subunits relative to promoter DNA and to the RNA transcript will be determined at defined steps in transcription using DNA-protein and RNA-protein photochemical cross- linking methods. In addition, the interactions between RNAP and the transcription initiation factors TBP and TFB will be investigated by protein-protein footprinting and protein cross-linking methods. Finally, the function of a putative novel transcription factor, homologous to TFB, will be investigated through binding and transcription assays with a variety of P. furiosus promoters. These experiments should provide an excellent overview of the structure of the archaeal transcription apparatus, and will provide a foundation for studying the regulation of transcription in this biological domain. Because of the current scarcity of basic knowledge concerning archaeal biology, the experiments in this proposal are very likely to result in discovery and to yield novel observations. In addition, the homology between archaeal and eucaryal transcription implies that conclusions from these experiments will be directly applicable to the study of eucaryal transcription. Therefore, this work may provide health benefits arising from applications to human inherited or acquired disease that originate from aberrant gene transcription. With a better understanding of eucaryal transcriptional mechanism, points of leverage for disease control may be identified and exploited.
