Empirically Defining Gene Architecture and Expression of M. Tuberculosis

 描述（由申请人提供）：分枝杆菌疾病，主要是结核病，每年造成近200万人死亡。无效的疫苗，以及多重耐药和极端耐药的M。结核病加剧了这一慢性全球危机。对基因组尺度方法在M.结核病（Mtb）为生物学见解提供了一个新的和强大的工具。任何基因组学方法的基础都是精确注释的基因组，特别是知道活性基因及其编码的蛋白质的精确边界。注释管道与具有显著核苷酸偏差和非典型基因结构的基因组斗争。因此，帮助基础和临床研究人员导航Mtb基因组的注释通常是不准确的。我们将RNA-seq与核糖体分析（Ribo-seq）相结合，以经验性地确定基因组规模上的转录和翻译起始位点，减少对计算基因预测的依赖。我们对模式分枝杆菌M. smeglavin等的研究表明，约三分之一的转录起始位点也是翻译起始位点，表明一大群基因没有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，经常导致它们的错误注释。我们的经验数据还鉴定了>300个未注释的编码在注释基因上游的肽。作为一类，这些上游肽尚未得到很好的研究，但有顺式调节或其他功能作用的先例。这些数据使得对M.这将增加所有后续工作的精度和信心预测基因组注释。在这里，我们建议使用相同的综合方法重新注释结核分枝杆菌基因组。结核分枝杆菌将在标准实验室条件下培养，并在模拟体内环境的条件下培养，以使尽可能多的基因表达最大化，特别是那些与发病机制最相关的基因。我们的Ribo-seq分析将在以未满足的精度和灵敏度映射蛋白质N-末端和上游肽方面提供特别的信息。我们希望能够鉴定出数百个新的基因起始点、新的肽（有前导的和无前导的）和非编码RNA，这些将为分枝杆菌群落提供重要信息。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翻译起始，肽组学，翻译调控和RNA聚合酶或核糖体持续合成能力的序列背景调节等新兴研究领域提供新的生物学见解。这些未充分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结核分枝杆菌生物学的知识，为治疗靶点提供新的见解。这项拟议的R21应用尖端工具来生成数据，确保提供经验支持的结核分枝杆菌基因组重新注释，这将对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提供种子。

 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.