Characterization of the Abundant Small Proteome of Mycobacteria

分枝杆菌丰富的小蛋白质组的表征

基本信息

批准号：
8949153
负责人：
KEITH M DERBYSHIRE
金额：
$ 23.62万
依托单位：
WADSWORTH CENTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8949153
关键词：
5&apos Untranslated Regions Amino Acids Animal Model Appearance Architecture Bacteria Bacterial Genes Biological Biology Cell division Cell physiology Cells Characteristics Chronic Clinical Codon Nucleotides Communities Coupled Coupling Cysteine Cytoplasm Data Disease Drug resistance Enzymes Escherichia coli Future Gene Expression Gene Expression Regulation Genes Genetic Transcription Genome Genomics Genus Mycobacterium Knowledge Laboratories Maps Mass Spectrum Analysis Measures Membrane Methodology Molecular Genetics Molecular Profiling Multi-Drug Resistance Mutation Mycobacterium smegmatis Mycobacterium tuberculosis Open Reading Frames Operon Pathway interactions Pharmaceutical Preparations Play Preparation Proteins Proteome Proteomics Public Health RNA Reading Reading Frames Regulation Reporter Resolution Resources Ribosomes Role Seeds Signal Transduction Single Nucleotide Polymorphism Map Site Technology Testing Transcription Initiation Transcription Initiation Site Translating Translation Initiation Translations Transmembrane Transport Tuberculosis Vaccines Work design field study genetic approach genetic makeup genome annotation genome-wide global health innovation insight intercellular communication killings liquid chromatography mass spectrometry mycobacterial novel pathogen protein folding public health relevance research study resistant strain tool transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): Mycobacterial disease, primarily tuberculosis, kills nearly two million people annually. Ineffective vaccines, as well as multi-drug and extremely-drug resistant strains of Mycobacterium tuberculosis, exacerbate this chronic global crisis. The design of new efficacious drugs necessitates a fundamental understanding of the biology of the bacterium and its genetic make-up. In turn, the successful application of genomic sequence information requires accurate gene annotation and a comprehensive knowledge of gene architecture and expression profiles. We have empirically determined transcription and translation initiation sites on a genome scale, using RNA- seq and ribosomal profiling (ribo-seq). This work has revealed two novel characteristics of mycobacterial gene architecture: one-third of transcription start sites lack a 5' UTR (leaderless genes lacking a Shine- Dalgarno sequence), and the presence of many hundreds of small open-reading-reading frames encoding small proteins of less than 50 amino acids (sproteins). To date, sproteins have been largely overlooked in bacteria, as they are hard to detect by traditional methodologies or annotation pipelines. Moreover, sproteins represent a completely unexplored class of proteins in mycobacteria, and are likely to have a significant impact on cell physiology. Importantly, our preliminary studies indicate that sproteins are present in large numbers in both the fast-growing model-organism, Mycobacterium smegmatis, and the slow- growing pathogen, M. tuberculosis, Here, we propose to generate the first comprehensive, experimentally validated small proteome for both slow- and fast- growing Mycobacteria. This will be achieved by combining state-of-the art sproteome mass spectrometry approaches with ribo-seq and transcription start site mapping. Together, these approaches will provide an empirically defined, high-confidence data resource for the mycobacterial community. In addition, we will define the mechanism of action for a subset of the sproteins that we hypothesize act in cis to regulate downstream genes. This proposal is highly innovative as it focuses on the discovery of an entirely unexpected class of abundant proteins that has escaped scientific scrutiny for any bacterium. The application of high-throughput, cutting- edge tools to facilitate this analysis will provide a comprehensive overview of the mycobacterial sproteome, while also providing mechanistic insight into potential functions of sproteins. Thus, we anticipate both an immediate and long-term impact on the mycobacterial field, providing new biological insights that will seed multiple emerging fields of study, while expanding our knowledge of gene architecture and regulation for all bacteria.

描述（由申请人提供）：分枝杆菌疾病，主要是结核病，每年导致近 200 万人死亡，而且结核分枝杆菌的多药和极度耐药菌株，加剧了这一慢性全球危机。药物需要对细菌的生物学及其基因组成有基本的了解，而基因组序列信息的成功应用需要准确的基因。我们利用 RNA-seq 和核糖体分析 (ribo-seq) 在基因组规模上凭经验确定了转录和翻译起始位点。这项工作揭示了分枝杆菌基因结构的两个新特征。：三分之一的转录起始位点缺乏 5' UTR（缺乏 Shine-Dalgarno 序列的无前导基因），并且存在数百个编码小于迄今为止，s 蛋白在细菌中基本上被忽视，因为它们很难通过传统方法或注释管道检测到。此外，s 蛋白代表了分枝杆菌中完全未被探索的一类蛋白质，并且可能具有重要的是，我们的初步研究表明，s蛋白在快速生长的模式生物分枝杆菌中大量存在。在这里，我们建议为缓慢和快速生长的分枝杆菌生成第一个全面的、经过实验验证的小蛋白质组，这将通过结合最先进的技术来实现。蛋白质组质谱方法与核糖测序和转录起始位点定位相结合，这些方法将为分枝杆菌群落提供经验定义的高可信度数据资源。我们将定义我们所追求的顺式作用以调节下游基因的 s 蛋白子集的作用机制。该提案具有高度创新性，因为它的重点是发现一类完全出乎意料的丰富蛋白质，这些蛋白质已经逃脱了任何科学审查。应用高通量、尖端的工具来促进这种分析将提供对分枝杆菌蛋白质组的全面概述，同时还提供对 s 蛋白潜在功能的机制洞察。对分枝杆菌领域产生长期影响，提供新的生物学见解，为多个新兴研究领域播种，同时扩展我们对所有细菌的基因结构和调控的知识。