RUI: Transcription Factor Discovery in the Extremophile Thermus Thermophilus

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With this award, the Genetic Mechanisms Cluster of the Division of Molecular and Cellular Biosciences: Biological Sciences Directorate is funding Dr. Michael Van Dyke from Kennesaw State University to identify orphan transcription factors and their potential biological roles. Transcription factors are the primary regulators of gene expression in most organisms, controlling those genes that are turned on or off in response to intrinsic factors or external cues. Presently, the targets of most transcription factors are not known. However, using a novel selection method that allows the screening of trillions of DNA sequences, it is now possible to go from putative transcription factor to understanding its biological regulatory roles in relatively short order. This project will explore potential transcription factors in Thermus thermophilus, a model extremophile organism whose proteins are widely used in many aspects of biotechnology. Knowledge derived from this project will be applicable to a host of industries, ranging from agribusiness to waste treatment. In addition, this project will train a mix of undergraduate and graduate (MS) students in the biochemistry of protein-DNA interactions as well as bioinformatics and regulon development, thereby providing future scientists in these fields. This research project will be undertaken to better understand transcriptional regulatory networks in the model extremophile T. thermophilus HB8. Binding specificity of putative transcription factors will be determined by the combinatorial approach Restriction Endonuclease Protection, Selection, and Amplification, followed by massively parallel semiconductor sequencing and Multiple Expectation Maximization for Motif Elicitation analysis. Potential binding sites will then be mapped to the Thermus thermophilus HB8 genome using Find Individual Motif Occurrences and functionally validated both in vitro (electrophoretic mobility shift assays) and in vivo (quantitative Polymerase Chain Reactions). Information from this study will provide a better understanding of regulons within a model eubacterial extremophile and will complement ongoing studies, for example, the Structural-Biological Whole Cell Project, which seeks to understand the molecular constituents of life at an atomic level. Methods developed by this project should also be generally applicable to characterizing orphan transcription factors and eliciting regulons in other organisms.