Small Proteins and Epitranscriptomic Factors: Emerging Mechanisms in Bacterial Gene Regulation

小蛋白质和表观转录因子：细菌基因调控的新兴机制

• 批准号: 10501172
• 负责人: Srujana Samhita Yadavalli
• 金额: $ 39.08万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10501172
• 关键词: Affect; Amino Acids; Anabolism; Antibiotics; Antimicrobial Resistance; Bacteria; Bacterial Genes; Biochemical Pathway; Biology; Cell Survival; Cell division; Cell physiology; Environment; Enzymes; Escherichia coli; Eukaryota; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Growth and Development function; Mass Spectrum Analysis; Measures; Metabolism; Methods; Modification; Multiple Bacterial Drug Resistance; Nucleoside Q; Open Reading Frames; Phosphotransferases; Physiology; Play; Prokaryotic Cells; Proteins; Proteomics; RNA; Regulation; Regulator Genes; Regulatory Pathway; Research; Role; Signal Transduction; Site; Stress; System; Textbooks; Therapeutic; Therapeutic Intervention; Transfer RNA; Translations; Work; antimicrobial peptide; attenuation; base; biochemical tools; biological adaptation to stress; crosslink; design; epitranscriptomics; experience; gene repression; genome annotation; in vivo; insight; link protein; novel; programs; response; ribosome profiling; sensor; stressor; therapeutically effective; transcription factor

PROJECT SUMMARY Bacteria use a diverse set of gene regulatory mechanisms to successfully adapt to ever-changing environments. While some regulatory pathways for gene expression such as activation or repression by transcription factors, and transcription/translation attenuation systems are well-characterized text-book examples, others are just coming into prominence. My research program is focused on understanding two distinct bacterial gene regulatory mechanisms: (i) small protein regulators, less than 50 amino acids long, and (ii) epitranscriptomic proteins that link RNA modifications and translation to metabolism and stress response. Small proteins are directly encoded by short open reading frames, which were missed in initial genome annotations due to the preset size cut-offs for gene size. This group of proteins are increasingly shown to play significant roles in fundamental cellular processes such as cell division, growth and development, modulation of transport and signaling. Despite the advances in small protein discovery, there has been little progress in functional characterization of these new- found proteins. To tackle the major challenge in this emerging field, Theme 1 will focus on the identification and functional characterization of small proteins involved in bacterial stress responses. We will: (i) modify and develop ribosome-profiling methods – Ribo-RET and Ribo-RET-PUR – to measure translation rates and identify condition-specific small proteins, (ii) develop an in vivo site-specific photo-cross-linking and a proteomics-based approach – SPICE-MS (Small Protein Interactions via Crosslinked Ensemble Mass Spectrometry) – tailored to capture small protein targets. Based on my previous experience with elucidating the interactions between a small protein MgrB and its target PhoQ sensor kinase, we will systematically characterize the functions of small proteins and their associated targets identified here using genetic and biochemical tools. Functional and mechanistic analyses of these small proteins will be useful in designing novel antibiotics and therapeutics. In addition, the methods developed here will be broadly applicable to small proteins from other prokaryotes as well as eukaryotes. In theme 2, we will focus on studying epitranscriptomic enzymes and their regulatory roles. Specifically, we will investigate role of QueE – an enzyme involved in the biosynthesis of a ubiquitous RNA modification called queuosine – in bacterial cell division during antimicrobial peptide stress. RNA modifications and the related machinery are modulated in response to different cellular stressors, and little is known about how this regulation affects cell physiology. We will investigate the mechanisms by which regulation of this tRNA modification enzyme, QueE affects cell division, translation and metabolism during antimicrobial peptide stress response in E. coli. Together, our work will advance the fields of small protein biology and epitranscriptomics by (a) identifying and characterizing small proteins involved in stress responses, and (b) depicting how epitranscriptomic enzymes act as nodes connecting translation to cellular metabolism and physiology, respectively.

项目摘要 细菌使用一组潜水员的基因调节机制来成功适应不断变化的环境。 尽管某些基因表达的调节途径，例如转录因子激活或表达，但 和转录/翻译衰减系统是特定的教科书示例，其他只是 突出。我的研究计划的重点是理解两个不同的细菌基因调节 机制：（i）小蛋白质调节剂，小于50氨基酸的长度，以及（ii）表转录蛋白 将RNA修饰和转化与代谢和压力反应联系起来。小蛋白直接编码 通过简短的开放式阅读框 用于基因大小。这组蛋白质越来越被证明在基本细胞中起着重要作用 诸如细胞分裂，生长和发育，转运和信号传导的调节等过程。尽管有 在小蛋白质发现的进步中，这些新的 - 找到蛋白质。为了应对这个新兴领域的主要挑战，主题1将重点放在身份证明上 参与细菌应激反应的小蛋白的功能表征。我们将：（i）修改和 开发核糖体促进方法 - 肋骨和核糖ret-pur-以测量翻译率并识别 条件特异性的小蛋白质，（ii）开发体内特定于特定于特定的光合链接和基于蛋白质组学的链接 方法 - 香料-MS（通过交联集合质谱法进行小蛋白质相互作用） - 量身定制 捕获小蛋白质靶标。根据我以前阐明小型相互作用的经验 蛋白质MGRB及其靶标PHOQ传感器激酶，我们将系统地表征小的功能 蛋白质及其相关靶标在此使用遗传和生化工具确定。功能和 这些小蛋白质的机械分析将有助于设计新型的抗生素和治疗。在 此外，此处开发的方法也将广泛适用于其他原核生物的小蛋白质 作为真核生物。在主题2中，我们将专注于研究同意酶及其调节作用。 具体而言，我们将研究QUEE的作用 - 涉及无处不在RNA的生物合成的酶 修饰称为Queuosine - 在抗菌胡椒应激期间细菌细胞分裂中的修饰。 RNA修饰 并且相关的机械是根据不同的细胞应激源调节的，并且对如何如何了解 该调节会影响细胞生理。我们将研究该tRNA调节的机制 修饰酶，QUEE会影响抗菌胡椒应激期间细胞分裂，翻译和代谢 大肠杆菌的反应。我们的工作一起将通过 （a）识别和表征与压力反应有关的小蛋白质，以及（b）描绘 同源酶充当连接到细胞代谢和生理学的节点， 分别。