This award funds an investigation of transcription factors and their potential biological roles in an extremophile bacterium that survives in very high temperatures that would be lethal to plants and animals. Bacterial transcription factors are proteins that bind to DNA and turn nearby genes on or off in response to environmental or internal cues. Regulation is essential so that the bacteria can survive stress and reproduce as efficiently as possible. Presently, the DNA binding sites for most bacterial transcription factors are unknown. The lab has developed a novel selection method that can be conducted in a test tube and allows them to screen billions of DNA sequences to find ones that a transcription factor can bind. Having identified those sequences it is possible to find those sequences in the genome sequence of the bacterium and then infer a likely biological role for the transcription factor. This project aims to identify the DNA binding sites for transcription factors in the extremophile Thermus thermophilus, a model organism whose proteins are widely used in biotechnology and biomanufacturing. This project has implications for many industries, ranging from agribusiness and medical testing to consumer products and waste treatment. These applications could be improved through better understanding gene regulation in such extremophiles. This project will also train both undergraduate and master’s students in the biochemistry and bioinformatics of protein-DNA interactions, thereby providing future scientists with expertise in these fields.
This research project seeks to better understand transcriptional regulatory networks in the model extremophile T. thermophilus HB8. The binding specificity of putative transcription factors will be determined using the iterative selection method 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 in vitro (electrophoretic mobility shift assays, biolayer interferometry) and in vivo (quantitative Polymerase Chain Reactions, microarray data). Information from this study will provide a better understanding of regulons within a model bacterial extremophile. They will also contribute to ongoing collaborative projects, for example, the Structural-Biological Whole Cell Project, which seeks to understand the molecular constituents of life at an atomic level. Finally, this project's methods should broadly apply to characterizing orphan transcription factors and defining regulons in many other organisms.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
