Coordination Funds

现代基因组学和转录组学技术与系统的全基因组方法相结合，揭示了原核生物中意想不到的基因组复杂性。除了编码较大蛋白质和非编码RNA的基因外，在过去十年中，全球方法已经在许多原核基因组中发现了大量隐藏的含有短开放阅读框（sORF）的小基因。这些sORF通常编码长度小于50个氨基酸的蛋白质，并且由于自动基因注释工具中过于严格的假设，通常在自动基因预测的基因组注释中被遗漏。此外，由于技术限制，这些小蛋白很难检测。只有少数小蛋白质的特征在于其细胞功能，但表明它们可以在不同的功能场景中发挥重要作用，并具有广泛的功能。在该SPP的第一个供资阶段，已经实施和建立了新技术，这些技术能够在全基因组方法中对小蛋白进行全球分析-复杂的生物信息学预测、肽组学和核糖体分析-作为平台，并应用于SPP中研究的几种微生物。个别项目的成果主要包括主要通过全基因组筛选，然后进行第一次功能分析，鉴定和验证几种模式生物中的小蛋白质。这第二个资助阶段的总体目标更加侧重于阐明已鉴定的小蛋白及其相互作用伙伴的详细功能特征，以及研究调控和作用的潜在分子机制。我们设想发现以前被忽视的原核生物的基因表达和组装/拆卸大复合物的调节原则。因此，我们将特别强调小蛋白作用的分子机制，并结合Z-项目中中心方法的持续进一步发展，以支持详细功能分析中的项目（例如，通过尺寸排阻色谱法进行复杂组成分析，然后进行LC-MS/MS，包括Ribo-seq分析中的特定起始/终止信息），除了NMR方面的专业知识外，FRET和HDX分析以及高端成像工具将通过个别项目提供给SPP。我们还将包括全基因组方法的比较评估。新实现的全基因组鉴定工具是交联蛋白质组学和细胞背景下小蛋白质的发现和功能注释。总的来说，我们相信，通过展示小蛋白的代表性新功能和机制，我们将揭示目前未知的独特和普遍的原核基因调控和大复合物的组装/拆卸原则。

Modern genomics and transcriptomics technologies combined with systematic genome-wide approaches have uncovered an unexpected genome complexity in prokaryotes. Besides genes encoding larger proteins and non-coding RNAs, global approaches have over the past decade discovered a wealth of hidden small genes containing short open reading frames (sORFs) in many prokaryotic genomes. These sORFs often encode proteins smaller than 50 amino acids in length and have been typically missed in genome annotations by automated gene predictions due to too strict assumptions in the automated gene annotation tools. Besides, those small proteins have been difficult to detect due to technical limitations. Only few small proteins have been characterized concerning their cellular function, however demonstrating that they can play important roles in different functional scenarios and have a broad range of function. New technologies, which enable the global profiling of small proteins in genome-wide approaches - sophisticated bioinformatics predictions, peptidomics and ribosome profiling - have been implemented and established as platforms within the first funding phase of this SPP and applied to several microorganisms studied in the SPP. Results of the individual projects included predominantly the identification and verification of small proteins in several model organisms mainly by genome-wide screens followed by first functional analyses. The overall aim of this second funding phase focuses more on elucidating the detailed functional characterization of the identified small proteins and their interaction partners as well as studying the underlying molecular mechanisms of regulation and action. We envisage to uncover previously overlooked prokaryotic principles in regulation of gene expression and assembly/disassembly of large complexes. Thus, we will lay particular emphasis on the molecular mechanisms of action of small proteins combined with continuous further development of the central methods within the Z-projects to support the projects also in detailed functional analysis (e.g. complex composition analysis by size exclusion chromatography followed by LC-MS/MS, include specific start/stop information in the Ribo-seq analysis), besides expertise in NMR-, FRET- and HDX-analysis, and high end imaging tools will be available for the SPP through individual projects. We will also include comparative evaluation of the genome-wide approaches. Newly implemented tools for genome-wide identification are cross-link proteomics and discovery and functional annotation of small proteins in the cellular context. Overall, we are confident that by demonstrating representative new functions and mechanisms for small proteins, we will uncover currently unknown unique and universal principles in prokaryotic gene regulation and assembly/disassembly of large complexes.