This research program deals with basic mechanisms for regulating activities of genes. Its significance for human health relates to the requirement for a broad and fundamental understanding of transcription mechanisms and their associated protein-nucleic acid interactions as the basis for precise and specifically targeted therapeutic interventions in gene expression that have important application in combating microbial infection, as well as inheritable, somatic mutation-based and other human disease. The proposed research analyzes transcription and transcriptional regulation in the archaea. The special significance of archaea for understanding universal aspects of transcription mechanism is based on the mosaic character of their informational systems: archaeal RNA polymerases and core transcription factors are organized along eukaryotic lines, and many archaea possess histones, another eukaryotic hallmark, while their transcriptional regulators are predominantly of bacterial type. It is this combination of elements commonly regarded as mutually exclusive that promises new insights into universal transcription mechanisms. The following principal lines of research on archaeal transcription, transcriptional regulators and chromatin are proposed: 1) Transcriptional activation and repression by the archaeal Lrp family protein Ptr2, with emphasis on activation through interaction with components of the transcription machinery other than TBP, and transcriptional repression by misrecruitment of the transcription apparatus. 2) The identity of the transcription activation domain of Ptr2, and of certain Ptr2 homologues, including those that have been analyzed previously only as repressers. 3) Specification of the surface domain on TATA-binding protein (TBP) that interacts with the transcription activation domain of Ptr2. 4) Determination of the structure of a Ptr2-TBP-DNA complex (in collaboration). 5) Genome-wide analysis of transcriptional activation by Ptr2 and Ptr1 (genomic microarray analysis of in vitro transcription). 6) Transcriptional activation in the context of archaeal chromatin-forming proteins, including histones, emphasizing the potential activation-reinforcing action of chromatin, anti-repressor action of Ptr2, and competition between Ptr2 and chromatin proteins for DMA binding. 7) Development of an in vitro transcription system for one of the genetically tractable mesophilic archaea, and analysis to determine whether particular individual components of the archaeal transcription apparatus and chromatin are essential for cell viability.
