NrdR and the Regulation of Ribonucleotide Reductase Genes in Escherichia Coli

在所有生物体中，DNA的生物合成（在细胞分裂或染色体修复过程中）需要四种构建块，统称为脱氧核糖核苷三磷酸（dNTPs）。从化学上讲，dNTPs（也称为脱氧核糖核苷酸）是四种相应的脱氧核糖核苷（脱氧鸟苷、脱氧腺苷、脱氧胞苷和胸苷）的磷酸化衍生物。dNTPs的生物合成完全依赖于一种酶（核糖核苷酸还原酶，RNR），该酶将核糖核苷酸还原为相应的脱氧核糖核苷酸。这个反应对于DNA的重新合成是必不可少的，因此RNR是几乎所有生物体中必不可少的酶。RNR的活性和编码该酶两个亚基的基因的表达受到严格调控。这些调节机制确保细胞提供四种dNTPs的平衡供应，并且dNTPs池与DNA合成和修复的需求相匹配。细菌通常会产生一种以上的RNR。例如，在模式细菌大肠杆菌中，有三种rnr: NrdAB需要分子氧，是有氧条件下生长所必需的；NrdDG不依赖氧，是厌氧生长所必需的；NrdEF在各种应激反应中表达，可能需要为DNA修复过程提供dNTPs。编码这三种酶的基因都受到一种被称为NrdR的转录抑制因子的负调控。人们相信（尽管没有证实）NrdR在高水平dNTPs的情况下会抑制rnr编码基因的表达。因此，NrdR可能是一个稳态反馈回路的关键组成部分，当细胞dNTP浓度高时，rnr的产生被下调。本项目将对大肠杆菌NrdR蛋白的作用机制进行研究。将测量NrdR对不同核苷酸和脱氧核苷酸的亲和力，并评估它们对NrdR dna结合活性的影响。NrdR的结构特性及其与rnr编码基因启动子区DNA形成的复合物将通过电子显微镜和x射线晶体学进行研究。探讨NrdR等因子对氧化应激对NrdEF表达的调控作用。总的来说，这些研究将使我们更深入地了解NrdR的机制，以及它在控制编码rnr的基因表达方面所起的作用。更广泛的影响该项目将进一步了解基本的生物学过程，即DNA单体前体的生物合成，这是染色体复制和细胞增殖的必要先决条件。该研究将涉及博士后、研究生和本科生研究人员的培训和教学。本科生将有机会直接参与实验室研究，通过参加有学分的研究课程，并通过参加研究所资助的研究机会项目。高中学生也将有机会参加暑期学期的研究机会项目。首席研究员参与旨在与高中教师和学生建立指导关系的项目，以及为来自代表性不足群体的微生物学家提供指导和研究经验的项目，并将在当地小学开展外展活动。研究结果将发表在国际同行评议的期刊上，并将在国家和国际会议上传播。结果也将酌情在非技术文献和网上发表，并将在达拉斯德州大学通讯办公室的帮助下向更广泛的社区传播。研究生和本科生将参加专业学会的会议，并将展示他们的研究成果的口头和海报交流。美国微生物学会德克萨斯州分会非常活跃，每年召开两次会议，为研究生和本科生的研究人员提供了一个有利的环境，让他们可以展示他们的成果，并与同行和其他学术科学家建立联系。鼓励研究生参与本科生和高中生的教学，在本科课程中担任助教，在实验室中担任导师。

In all living organisms, the biosynthesis of DNA (during cell division or chromosome repair) requires four building blocks, collectively called deoxyribonucleoside triphosphates (dNTPs). Chemically, the dNTPs (also called deoxyribonucleotides) are phosphorylated derivates of the four corresponding deoxyribonucleosides (deoxyguanosine, deoxyadenosine, deoxycytidine and thymidine). The biological synthesis of dNTPs is absolutely dependent on an enzyme (ribonucleotide reductase, RNR), which reduces ribonucleotides to the corresponding deoxyribonucleotides. This reaction is essential for the de novo synthesis of DNA, and so RNR is an essential enzyme in virtually all organisms. The activity of RNR and the expression of the genes encoding the two subunits of the enzyme are tightly regulated. These regulatory mechanisms ensure that the cell is provided with a balanced supply of the four dNTPs, and that the pool of dNTPs is matched to the requirements of DNA synthesis and repair. Bacteria typically produce more than one type of RNR. For example, in the model bacterial species Escherichia coli, there are three RNRs: NrdAB requires molecular oxygen and is essential for growth under aerobic conditions; NrdDG is oxygen-independent and essential for anaerobic growth; NrdEF is expressed in response to various stresses and may be required to supply dNTPs for DNA repair processes. The genes encoding these three enzymes are all subject to negative regulation by a transcriptional repressor designated NrdR. It is believed (though not proven) that NrdR represses expression of the RNR-encoding genes in response to high levels of dNTPs. Thus, NrdR is likely to be a key component of a homeostatic feedback loop, in which production of the RNRs is down-regulated when cellular dNTP concentrations are high. In this project, the mechanism of the E. coli NrdR protein will be investigated. The affinity of NrdR for different nucleotides and deoxynucleotides will be measured, and their effects on the DNA-binding activity of NrdR evaluated. Structural properties of NrdR, and the complex that it forms with DNA from the promoter regions of the RNR-encoding genes will be investigated by electron microscopy and by X-ray crystallography. The contribution that NrdR and other factors make to the regulation of expression of NrdEF by oxidative stress will also be explored. Collectively, these studies will lead to a deeper understanding of the mechanism of NrdR, and the role that it plays in controlling the expression of the genes encoding RNRs.Broader impactsThe project will further understanding of a fundamental biological process, that is the biosynthesis of the monomeric precursors of DNA, an essential prerequisite to chromosome replication and cell proliferation. The research will involve the training and teaching of post-doctoral, graduate and undergraduate researchers. Undergraduates will have opportunities to participate directly in laboratory research, by taking credit-bearing research courses, and through participation in Institute-funded research opportunity programs. There will also be opportunities for high school students participating in summer semester research opportunity programs. The principal investigator participates in programs aimed at establishing mentoring relationships with high school teachers and students, and programs that mentor and provide research experiences for microbiologists from underrepresented groups, and will develop outreach activities in a local elementary school. Results of the research will be published in international peer-reviewed journals, and will be disseminated at national and international conferences. As appropriate, results will also be published in non-technical literature and online, and will be disseminated to the broader community with the help of the UT Dallas Office of Communications. Graduate and undergraduate students will attend meetings of professional societies, and will present oral and poster communications of the results of their research. The Texas Branch of the American Society for Microbiology is extremely active, meets twice a year, and provides a supportive environment in which graduate and undergraduate researchers can present their results and network with peers and other academic scientists. Graduate students will be encouraged to participate in the teaching of undergraduates and high school students, by acting as Teaching Assistants in undergraduate courses and as mentors in the lab.