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

项目总结 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/翻译衰减系统是很好地描述教科书的例子，其他只是 崭露头角。我的研究项目专注于了解两种不同的细菌基因调控 作用机制：(I)小于50个氨基酸的小蛋白调节剂，以及(Ii)表位转录蛋白 将RNA修饰和翻译与新陈代谢和应激反应联系起来。小分子蛋白质被直接编码 通过短的开放阅读框，其在初始基因组注释中由于预设的大小截止点而被遗漏 基因大小。这组蛋白质越来越多地被证明在基础细胞中扮演着重要的角色 细胞分裂、生长发育、运输调节和信号传递等过程。尽管 在小蛋白发现方面的进展，但对这些新的-蛋白质的功能鉴定进展甚微。 发现了蛋白质。为了应对这一新兴领域的主要挑战，主题1将集中于确定和 参与细菌应激反应的小蛋白的功能特征。我们将：(I)修改和 开发核糖体图谱分析方法--Ribo-RET和Ribo-RET-Pur-以测量翻译率并识别 条件特异性小蛋白，(Ii)开发体内部位特异性光交联和基于蛋白质组学的 方法-SPICE-MS(通过交联系谱进行小蛋白质相互作用)-量身定做 捕获小的蛋白质靶标。根据我以前的经验，我解释了一个小的 蛋白MgrB及其靶向的PhoQ感受器激酶，我们将系统地表征小分子的功能 使用遗传和生化工具确定的蛋白质及其相关靶标。功能和 对这些小蛋白质的机理分析将有助于设计新的抗生素和治疗方法。在……里面 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。 作为真核生物。在主题2中，我们将重点研究表位转录酶及其调节作用。 具体地说，我们将研究Quee的作用--一种参与无处不在的rna生物合成的酶。 在抗菌肽应激过程中，细菌细胞分裂过程中的修饰被称为队列蛋白。RNA修饰 而相关的机制是根据不同的细胞应激源进行调节的，人们对它们是如何调节的知之甚少。 这种调节影响细胞生理学。我们将研究调节这种tRNA的机制 修饰酶QUEE在抗菌肽应激过程中影响细胞分裂、翻译和代谢 在大肠杆菌中的反应。我们的工作将共同推动小蛋白生物学和表位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，以及(B)描述如何 表位转录酶作为连接翻译与细胞新陈代谢和生理的节点， 分别进行了分析。

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.