RUI: Transcription Factor Discovery in the Extremophile Thermus Thermophilus

凭借这一奖项，分子和细胞生物科学部门的遗传机制集群：生物科学理事会资助肯纳索州立大学的迈克尔·范·戴克博士识别孤儿转录因子及其潜在的生物学作用。在大多数生物体中，转录因子是基因表达的主要调节因子，控制那些响应内在因素或外部提示而开启或关闭的基因。目前，大多数转录因子的靶标尚不清楚。然而，使用一种新的选择方法，允许筛选数万亿个DNA序列，现在有可能在相对较短的顺序内从假定的转录因子到了解其生物调节作用。该项目将探索嗜热菌的潜在转录因子，这是一种极端微生物的模式生物，其蛋白质被广泛应用于生物技术的许多方面。从该项目获得的知识将适用于许多行业，从农业综合企业到废物处理。此外，该项目将在蛋白质-DNA相互作用的生物化学以及生物信息学和调节子开发方面培训一批本科生和研究生，从而为这些领域的未来科学家提供培训。开展这一研究项目的目的是为了更好地了解极端嗜热菌HB8模型中的转录调控网络。推测的转录因子的结合特异性将通过组合方法确定，限制性内切酶保护，选择和放大，然后大规模并行半导体测序和多期望最大化基序激发分析。然后，使用查找单个基序的方法将潜在的结合位点映射到Thermus thermophilus HB8基因组，并在体外(凝胶迁移率改变分析)和体内(定量聚合酶链式反应)进行功能验证。这项研究提供的信息将有助于更好地了解模型真细菌极端细菌内的调节因子，并将补充正在进行的研究，例如结构-生物全细胞项目，该项目寻求在原子水平上了解生命的分子组成。该项目开发的方法也应该普遍适用于表征孤儿转录因子和在其他生物体中引发调控。

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.