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.

项目概要 细菌利用多种基因调控机制来成功适应不断变化的环境。 虽然基因表达的一些调控途径如转录因子的激活或抑制， 转录/翻译衰减系统是教科书上典型的例子，其他的只是 逐渐崭露头角。我的研究项目重点是了解两种不同的细菌基因调控 机制：(i) 小蛋白调节因子，长度少于 50 个氨基酸，以及 (ii) 表观转录组蛋白 将 RNA 修饰和翻译与新陈代谢和应激反应联系起来。小蛋白质直接编码 通过短的开放阅读框，由于预设的大小截止，这些框架在初始基因组注释中被遗漏 对于基因大小。这组蛋白质越来越多地被证明在基本细胞中发挥着重要作用 细胞分裂、生长和发育、运输和信号传导调节等过程。尽管 尽管小蛋白质发现取得了进展，但这些新蛋白质的功能表征却进展甚微。 发现了蛋白质。为了应对这一新兴领域的主要挑战，主题 1 将重点关注识别和 参与细菌应激反应的小蛋白的功能表征。我们将： (i) 修改并 开发核糖体分析方法 – Ribo-RET 和 Ribo-RET-PUR – 测量翻译率并识别 条件特异性小蛋白，(ii) 开发体内位点特异性光交联和基于蛋白质组学的 方法 – SPICE-MS（通过交联集成质谱法的小蛋白质相互作用） – 量身定制 捕获小蛋白质目标。根据我之前阐明小事物之间相互作用的经验 蛋白 MgrB 及其靶标 PhoQ 传感器激酶，我们将系统地表征小分子的功能 使用遗传和生化工具在此确定蛋白质及其相关靶点。功能性和 这些小蛋白质的机制分析将有助于设计新型抗生素和治疗方法。在 此外，这里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，我们将重点研究表观转录酶及其调控作用。 具体来说，我们将研究 QueE 的作用——一种参与普遍存在的 RNA 生物合成的酶 称为queuosine的修饰——在抗菌肽应激期间细菌细胞分裂中。 RNA修饰 相关机制会根据不同的细胞应激源进行调节，但人们对如何调节知之甚少 这种调节影响细胞生理学。我们将研究该 tRNA 的调节机制 修饰酶 QueE 在抗菌肽应激过程中影响细胞分裂、翻译和代谢 大肠杆菌中的反应。我们的工作将共同推进小蛋白质生物学和表观转录组学领域的发展 (a) 识别和表征参与应激反应的小蛋白质，以及 (b) 描述如何 表观转录酶充当连接翻译与细胞代谢和生理学的节点， 分别。