Empirically Defining Gene Architecture and Expression of M. Tuberculosis

结核分枝杆菌基因结构和表达的实证定义

• 批准号: 8868643
• 负责人: KEITH M DERBYSHIRE
• 金额: $ 18.14万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8868643
• 关键词: 5' Untranslated Regions; Address; Animal Model; Appearance; Architecture; Area; Biological; Biological Assay; Biology; Chronic; Clinical; Code; Communities; Coupled; DNA-Directed RNA Polymerase; Data; Data Set; Disease; Drug Targeting; Drug resistance; Environment; Foundations; Frequencies; Future; Gene Expression; Gene Proteins; Gene Structure; Genes; Genetic; Genome; Genomic approach; Genomics; Genus Mycobacterium; Growth; Hypoxia; Knowledge; Laboratories; Maps; Measures; Messenger RNA; Methods; Modeling; Multi-Drug Resistance; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nucleotides; Pathogenesis; Peptides; Pharmaceutical Preparations; Physiological; Play; Proteins; RNA; Regulation; Research; Research Personnel; Resolution; Ribosomes; Role; Seeds; Site; Surveys; Transcription Initiation; Transcription Initiation Site; Translating; Translation Initiation; Translational Regulation; Translations; Tuberculosis; Untranslated RNA; Vaccines; Work; field study; genome annotation; genome-wide; global health; in vivo; innovation; insight; killings; mycobacterial; novel; pathogen; public health relevance; research study; resistant strain; therapeutic target; tool; transcriptome sequencing

 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 M. tuberculosis, exacerbate this chronic global crisis. The application o genome-scale approaches to M. tuberculosis (Mtb) provides a new and powerful tool for biological insights. The foundation of any genomic approach is an accurately annotated genome, particularly knowing the precise boundaries of active genes and the proteins they encode. Annotation pipelines struggle with genomes that have significant nucleotide bias and atypical gene structures. As such, the annotations that help basic and clinical researchers navigate the Mtb genome are often inaccurate. We have integrated RNA-seq with ribosomal profiling (Ribo-seq) to empirically determine transcription and translation initiation sites on a genome scale, reducing the reliance on computational gene predictions. Our survey of the model mycobacterium, M. smegmatis, showed that about one-third of transcription start sites were also translation initiation sites, indicating a large group of genes without a 5' UTR. These leaderless genes lack a Shine-Dalgarno sequence, the traditional landmark that helps to predict translation initiation sites, frequently contributing to their misannotation. Our empirical data alo identified >300 unannotated peptides encoded upstream of annotated genes. As a class, these upstream peptides have not been well studied, yet there are precedents for cis-regulatory or other functional roles. These data allowed a much more accurate re- annotation of the M. smegmatis genome, which will augment the precision and confidence of all subsequent work predicated on genome annotations. Here, we propose to re-annotate the Mtb genome using the same integrative approach. Mtb will be cultured under standard laboratory conditions, and under conditions that simulate in vivo environments to maximize the expression of as many genes as possible, especially those most relevant to pathogenesis. Our Ribo-seq analyses will be particularly informative in mapping protein N-termini and upstream peptides with unmet precision and sensitivity. We expect to identify many hundreds of new gene starts, novel peptides (both leadered and leaderless) and non-coding RNAs that will provide vital information to the mycobacterial community. While our data will be instrumental in defining gene boundaries in the Mtb genome, they will also offer new biological insights into the fledgling research areas of leaderless translation initiation, peptidomics, translational regulation, and sequence context modulation of RNA polymerase or ribosome processivity. Each of these understudied topics will expand our knowledge of gene architecture and regulation, and the biology of Mtb, providing new insights for therapeutic targets. This proposed R21 applies cutting-edge tools to generate data assured to provide an empirically supported re-annotation of the Mtb genome that will have an immediate and protracted impact in field, while providing new biological insights that will seed multiple emerging fields of study.

 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，以及结核分枝杆菌的多药和极度耐药菌株，加剧了这一长期的全球危机。结核分枝杆菌 (Mtb) 的大规模方法为生物学见解提供了一种新的强大工具，任何基因组方法的基础都是准确注释的基因组，特别是了解活性的精确边界。基因及其编码的蛋白质的注释管道与具有显着核苷酸偏差和非典型基因结构的基因组相矛盾，因此，帮助基础和临床研究人员导航结核分枝杆菌基因组的注释通常是不准确的。 （Ribo-seq）在基因组规模上凭经验确定转录和翻译起始位点，减少对计算基因预测的依赖，我们对模型分枝杆菌，耻垢分枝杆菌的调查表明。大约三分之一的转录起始位点也是翻译起始位点，表明大量基因没有 5' UTR，这些无前导基因缺乏 Shine-Dalgarno 序列，这是有助于预测翻译起始位点的传统标志，经常发挥作用。我们的经验数据还发现了超过 300 个编码注释基因上游的未注释肽，这些上游肽尚未得到充分研究，但有顺式调节的先例。这些数据可以对耻垢分枝杆菌基因组进行更准确的重新注释，这将提高基于基因组注释的所有后续工作的精度和可信度。在这里，我们建议使用 Mtb 基因组重新注释。 Mtb 将在标准实验室条件下以及模拟体内环境的条件下进行培养，以最大限度地表达尽可能多的基因，尤其是与发病机制最相关的基因。分析将在以未满足的精度和灵敏度绘制蛋白质 N 末端和上游肽方面提供特别丰富的信息，我们期望识别数百个新的基因起始点、新的肽（有前导肽和无前导肽）和非编码 RNA，这些将为我们提供重要信息。虽然我们的数据将有助于确定结核分枝杆菌基因组中的基因边界，但它们也将为无领导者翻译起始、肽组学、翻译调控和序列等新兴研究领域提供新的生物学见解。这些正在研究的主题中的每一个都将扩展我们对基因结构和调控以及 Mtb 生物学的知识，为治疗目标提供新的见解。提供基于经验的 Mtb 基因组重新注释，这将在该领域产生直接和持久的影响，同时提供新的生物学见解，为多个新兴研究领域奠定基础。