Characterization of the Abundant Small Proteome of Mycobacteria

 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。无效的疫苗以及结核分枝杆菌的多药和极端耐药菌株加剧了这一慢性全球危机。设计新的有效药物需要对细菌的生物学及其遗传组成有基本的了解。反过来，基因组序列信息的成功应用需要准确的基因注释和基因结构和表达谱的全面知识。我们已经使用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），以及存在数百个编码小于50个氨基酸的小蛋白质（s蛋白质）的小开放阅读-阅读框架。迄今为止，细菌中的s蛋白在很大程度上被忽视了，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开发的一类蛋白质，并且可能对细胞生理学产生重大影响。重要的是，我们的初步研究表明，在快速生长的模式生物耻垢分枝杆菌和缓慢生长的病原体耻垢分枝杆菌中都存在大量的s蛋白。在这里，我们提出了第一个全面的，实验验证的小蛋白质组的缓慢和快速增长的分枝杆菌。这将通过将最先进的蛋白质组质谱方法与核糖测序和转录起始位点作图相结合来实现。总之，这些方法将为分枝杆菌群落提供经验定义的高置信度数据资源。此外，我们将定义的一个子集的s蛋白，我们假设顺式作用，以调节下游基因的作用机制。这一提议具有高度创新性，因为它专注于发现一种完全出乎意料的丰富蛋白质，这种蛋白质已经逃脱了任何细菌的科学审查。应用高通量、尖端的工具来促进这种分析将提供分枝杆菌s蛋白质组的全面概述，同时也提供对s蛋白质潜在功能的机理性洞察。因此，我们预计对分枝杆菌领域的直接和长期影响，提供新的生物学见解，将为多个新兴研究领域提供种子，同时扩大我们对所有细菌基因结构和调控的知识。

 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.