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RUI: Transcription Factor Discovery in the Extremophile Thermus Thermophilus

RUI：在嗜热栖热菌中发现转录因子

基本信息

批准号：
2041202
负责人：
Michael Van Dyke
金额：
$ 33万
依托单位：
Kennesaw State University Research and Service Foundation
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=2041202&HistoricalAwards=false
关键词：
RUI Transcription Factor Discovery Extremophile

项目摘要

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.

该奖项资助了一项对转录因子及其在极端细菌中的潜在生物学作用的研究，该细菌在对植物和动物致命的高温下存活。细菌转录因子是与DNA结合的蛋白质，并根据环境或内部线索打开或关闭附近的基因。调节是必不可少的，这样细菌才能在压力下生存并尽可能有效地繁殖。目前，大多数细菌转录因子的DNA结合位点是未知的。该实验室开发了一种新的选择方法，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序列。在鉴定了这些序列之后，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。该项目旨在确定极端嗜热菌Thermus thermophilus中转录因子的DNA结合位点，Thermus thermophilus是一种模式生物，其蛋白质广泛用于生物技术和生物制造。该项目对许多行业都有影响，从农业综合企业和医学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中的基因调控来改进。该项目还将对本科生和硕士生进行蛋白质-DNA相互作用的生物化学和生物信息学培训，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型极端嗜热菌T。嗜热菌HB 8。将使用迭代选择方法限制性核酸内切酶保护、选择和扩增，然后进行大规模平行半导体测序和多期望最大化基序激发分析，确定推定转录因子的结合特异性。然后，使用Find Individual Motif Occurrences将潜在结合位点定位到嗜热栖热菌HB 8基因组，并在体外（电泳迁移率变化试验，生物层干涉法）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了解调节子内的一个模型细菌极端微生物。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，该项目旨在了解原子水平上生命的分子组成。最后，该项目的方法应广泛适用于表征孤儿转录因子和定义调节子在许多其他organis.This奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。