Systematic Discovery and Analysis of Small Proteins and Small ORFs in Mycobacteria

在过去的几十年里，我们看到了分子生物学领域的诞生和成熟。最初，突变 基因是基因组探索的焦点。现在，整个基因组都被例行测序， 通过注释算法自动识别常驻基因。另外，蛋白质组学方法 制备用于质谱分析的全细胞提取物的蛋白水解肽。这一切成功都 方法对大基因有强烈的偏见：大基因是突变的常见目标，长期开放， 阅读框在基因组序列中很容易识别，大的蛋白质产生许多肽 用于质谱鉴定。这种无意的偏见也造成了我们的巨大差距， 了解分子生物学。 最近在真核生物和原核生物中的研究发现了大量的小基因或其 编码蛋白质。小分子蛋白质（被认为是50个氨基酸或更少）的数量与传统的 大型蛋白质，但只有少数被认为具有功能。该提案的目的是推动 这一新兴领域的发展，促进了小蛋白的发现和功能表征。我们 初步数据确定了明确定义顺式和反式功能的具体例子， 开放阅读框和小蛋白。这些早期的线索将得到落实， 任何新领域都需要扩展严格的研究框架。我们将测试一个额外的子集， 小蛋白的功能，我们预计将揭示这个训练集的每个成员的功能， 同时也建立了短开放阅读框和小蛋白的一般原则。 我们将在分枝杆菌中开发和应用我们的小蛋白方法。分枝杆菌提供许多 小蛋白研究的优势。最重要的是，它们在标准蛋白质中表达>1000种小蛋白质。 条件一个广泛的工具包，用于修改，培养和分析分枝杆菌，使他们非常 听话富含GC的基因组提供密码子偏好选择作为鉴定功能性小分子的一个标准。 proteins.此外，我们在标准实验室条件下对小基因/蛋白质功能的发现可能 直接提供对感染的生物学和发病机理的见解。该提案将 调查人员的补充专门知识，他们的持续合作已经提供了必要的 导致这一提议的基础。通过拟议的目标，我们将确定 编码分枝杆菌小蛋白，并开发一个优化的，小蛋白质组学管道， 应用于其他细菌、古细菌和真核生物。

The last few decades have seen the birth and maturation of the field of Molecular Biology. Initially, mutant genes were focal points of genome exploration. Now, entire genomes are routinely sequenced, and the resident genes are automatically identified by annotation algorithms. Alternatively, proteomic approaches prepare proteolytic peptides of whole-cell extracts for analysis by mass spectrometry. Each of these approaches are strongly biased for large genes: large genes are frequent targets for mutation, long-open- reading frames are easily discerned in genomic sequence, and large proteins generate many peptides for mass spectrometry identification. This unintended bias has also created a large gap in our understanding of molecular biology. Recent work in eukaryotes and prokaryotes alike have uncovered multitudes of small genes or their encoded proteins. The numbers of small proteins (considered as 50 aa or less) rival that of traditionally large proteins, yet only a handful have been ascribed a function. The goal of this proposal is to propel this nascent field forward by facilitating both small protein discovery and functional characterization. Our preliminary data identify specific examples that clearly define cis- and trans-classes of function for short- open-reading frames and small proteins. These early leads will be pursued to fruition, providing the framework for the expanded rigorous study needed in any new field. We will test an additional subset of small proteins for function, which we anticipate will reveal functions for each member of this training set, while also establishing general principles for short-open-reading frames and small proteins. We will develop and apply our small protein approaches in mycobacteria. Mycobacteria offer many advantages for small protein study. Foremost is that they express >1000 small proteins in standard conditions. An extensive toolkit for modifying, culturing, and analyzing mycobacteria makes them very tractable. A GC-rich genome provides codon bias selection as one criterion to identify functional small proteins. Moreover, our findings of small gene/protein function in standard laboratory conditions may directly provide insights into the biology and pathogenesis of infection. This proposal integrates the complementary expertise of investigators whose ongoing collaboration has already provided the requisite groundwork leading to this proposal. Through the proposed Aims, we will identify new functional roles of encoded mycobacterial small proteins and develop an optimized, small-proteomics pipeline for efficient application to other bacteria, archaea, and eukaryotes.