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.

项目总结