RUI: Transcription Factor Discovery in the Extremophile Thermus Thermophilus

该奖项资助了转录因子及其在极端微生物细菌中的潜在生物学作用的研究，这种细菌可以在对植物和动物致命的极高温度下生存。细菌转录因子是一种与DNA结合并根据环境或内部信号开启或关闭附近基因的蛋白质。调节是必不可少的，这样细菌才能在压力下生存，并尽可能高效地繁殖。目前，大多数细菌转录因子的DNA结合位点是未知的。该实验室已经开发出一种新的选择方法，可以在试管中进行，并允许他们筛选数十亿个DNA序列，以找到转录因子可以结合的DNA序列。确定了这些序列，就有可能在细菌的基因组序列中找到这些序列，然后推断转录因子可能的生物学作用。该项目旨在鉴定嗜热热菌转录因子的DNA结合位点，嗜热热菌是一种模式生物，其蛋白质广泛应用于生物技术和生物制造。这个项目对许多行业都有影响，从农业综合企业和医疗测试到消费品和废物处理。这些应用可以通过更好地了解这些极端微生物的基因调控来改进。该项目还将培养蛋白质- dna相互作用的生物化学和生物信息学方面的本科生和硕士生，从而为未来的科学家提供这些领域的专业知识。本研究项目旨在更好地了解模式嗜热嗜热菌HB8的转录调控网络。假设的转录因子的结合特异性将使用迭代选择方法限制性内切酶保护，选择和扩增来确定，然后进行大规模并行半导体测序和Motif Elicitation分析的多重期望最大化。潜在的结合位点将被映射到热嗜热菌HB8基因组，并在体外（电泳迁移率转移测定，生物层干涉测定）和体内（定量聚合酶链反应，微阵列数据）进行功能验证。从这项研究的信息将提供一个更好的理解模式细菌极端微生物的调控。他们还将为正在进行的合作项目做出贡献，例如，结构生物全细胞项目，该项目旨在从原子水平上理解生命的分子组成。最后，这个项目的方法应该广泛适用于孤儿转录因子的特征和定义许多其他生物的规则。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.