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

项目摘要 细菌使用一套不同的基因调控机制来成功地适应不断变化的环境。 而一些基因表达的调控途径如转录因子的激活或抑制， 和转录/翻译衰减系统是典型的教科书例子，其他的只是 变得突出。我的研究项目集中在了解两种不同的细菌基因调控 机制：（i）小蛋白调节剂，长度小于50个氨基酸，和（ii）表转录组蛋白， 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值， 基因大小这组蛋白质越来越多地显示出在基础细胞中发挥重要作用。 细胞分裂、生长和发育、运输和信号传导的调节等过程。尽管 尽管小蛋白质发现的进展，但这些新蛋白质的功能表征进展甚微， 发现蛋白质为了应对这一新兴领域的重大挑战，主题1将侧重于确定和 参与细菌应激反应的小蛋白的功能表征。我们将：（i）修改和 开发核糖体分析方法- Ribo-RET和Ribo-RET-PUR -以测量翻译速率并识别 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（通过交联包被质谱法的小蛋白相互作用）-专为 捕获小的蛋白质目标。根据我以前的经验，阐明了一个小的 蛋白MgrB及其靶点PhoQ传感器激酶，我们将系统地表征小分子的功能 蛋白质及其相关的目标确定这里使用遗传和生物化学工具。功能和 对这些小蛋白的机理分析将有助于设计新的抗生素和治疗剂。在 此外，本文所建立的方法也可广泛应用于其他原核生物的小分子蛋白质的研究 真核生物一样。主题二，我们将重点研究表转录组酶及其调控作用。 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 在抗菌肽应激过程中，细菌细胞分裂中的一种叫做肌苷的修饰。RNA修饰 以及相关的机制在不同的细胞应激源中被调节， 这种调节影响细胞生理学。我们将研究调节这种tRNA的机制， QueE修饰酶在抗菌肽应激过程中影响细胞分裂、翻译和代谢 响应E.杆菌我们的工作将共同推进小蛋白生物学和表观转录组学领域， (a)鉴定和表征参与应激反应的小蛋白，和（B）描述如何 表转录组酶作为连接翻译与细胞代谢和生理学的节点， 分别

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.