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

分枝杆菌中小蛋白和小 ORF 的系统发现和分析

• 批准号: 10452528
• 负责人: KEITH M DERBYSHIRE
• 金额: $ 54.7万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452528
• 关键词: 5' Untranslated Regions; Algorithms; Amino Acids; Archaea; Architecture; Bacteria; Biochemical; Bioinformatics; Biology; Birth; Cell Extracts; Codon Nucleotides; Collaborations; Coupling; Cryoelectron Microscopy; Cysteine; Data; Data Set; Detection; Eukaryota; Fruit; Gene Proteins; Genes; Genome; Genomics; Genus Mycobacterium; Goals; Infection; Instruction; Knowledge; Laboratories; Life; Mass Spectrum Analysis; Medical; Messenger RNA; Methods; Molecular Biology; Molecular Genetics; Mutation; Open Reading Frames; Operon; Organism; Pathogenesis; Pathogenicity; Peptides; Preparation; Prevalence; Prokaryotic Cells; Property; Proteins; Proteomics; Regulation; Research Personnel; Ribosomal Proteins; Ribosomes; Role; Selection Bias; Signal Transduction; Structure; Testing; Training; Translating; Translations; Work; attenuation; comparative; experimental study; genetic approach; genome annotation; genome-wide; improved; insight; member; mutant; mycobacterial; novel; protein function; response; ribosome profiling; whole genome

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.

在过去的几十年中，分子生物学领域的出生和成熟。最初，突变体 基因是基因组探索的焦点。现在，整个基因组常规测序，并且 居民基因通过注释算法自动鉴定。或者，蛋白质组学方法 制备全细胞提取物的蛋白水解肽，以通过质谱法分析。每个 对于大基因而言，方法有很大的偏见：大基因是突变的频繁靶标，长期开放 读取帧很容易以基因组序列辨别，并且大蛋白会产生许多肽 用于质谱识别。这种意外的偏见也在我们的 了解分子生物学。 真核生物和原核生物的最新工作都发现了许多小基因或其它们 编码的蛋白质。传统上，小蛋白质的数量（被认为是50 aa或以下）与 大型蛋白质，但只有少数属性归因于功能。该提议的目的是推动 通过促进小蛋白质发现和功能表征，这一新生场的前进。我们的 初步数据确定了特定示例，这些示例清楚地定义了短 - 开读框架和小蛋白质。这些早期的潜在客户将被实现，提供 在任何新领域都需要进行扩展的严格研究框架。我们将测试另一个子集 用于功能的小蛋白质，我们预计这将揭示该训练集的每个成员的功能， 同时还建立了用于短读框架和小蛋白质的一般原则。 我们将在分枝杆菌中开发并应用我们的小蛋白质方法。分枝杆菌提供了许多 小蛋白质研究的优势。最重要的是它们在标准中表达> 1000个小蛋白 状况。用于修改，培养和分析分枝杆菌的广泛工具包使它们非常 可处理。富含GC的基因组可提供密码子偏置选择作为识别功能小的一个标准 蛋白质。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 直接提供有关感染生物学和发病机理的见解。该提案整合了 调查人员的互补专业知识，他们的持续合作已经提供了必要的 基础工作导致该提议。通过拟议的目标，我们将确定 编码分枝杆菌小蛋白质，并开发出优化的小型蛋白质管道，以提高 应用于其他细菌，古细菌和真核生物。