Mechanism of Translation Initiation on Leaderless mRNAs

无领导者 mRNA 的翻译起始机制

• 批准号: 10593807
• 负责人: Alexey Petrov
• 金额: $ 22.17万
• 依托单位: AUBURN UNIVERSITY AT AUBURN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-10 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10593807
• 关键词: 5' Untranslated Regions; Address; Affect; Antibiotic Resistance; Archaea; Bacteria; Bacterial Physiology; Base Pairing; Binding; Biochemical; Cells; Codon Nucleotides; Dissection; Elements; Eukaryota; Fluorescence; Fluorescence Resonance Energy Transfer; Genus Mycobacterium; Guanosine Triphosphate; Hydrolysis; Initiator Codon; Initiator tRNA; Label; Messenger RNA; Methods; Microscopy; Modeling; Molecular; Molecular Conformation; Nature; Nucleotides; Paint; Pathway interactions; Peptide Initiation Factors; Play; Prevalence; Process; Protein Biosynthesis; Proteins; Proteome; Regulation; Ribosomal RNA; Ribosomes; Role; Structure; System; Testing; Textbooks; Time; Transfer RNA; Translating; Translation Initiation; Translations; analog; biological adaptation to stress; in vivo; interest; mRNA Expression; mRNA Translation; mycobacterial; negative affect; novel; recruit; single molecule; translation factor

SUMMARY Leaderless mRNAs lack a 5’ UTR and SD sequence. Such mRNAs are common in Bacteria and Archaea, with Mycobacteria being the most striking example, as about one-third of mycobacterial mRNAs are naturally leaderless. Leaderless translation is part of a stress response and adaptation mechanism in bacteria. In contrast to the well-understood Shine Dalgarno-containing mRNAs, very little is known about the initiation mechanism of leaderless mRNAs. Biochemical, experimental, and systematic analysis of leaderless mRNA expression suggested that leaderless mRNAs are translated and regulated via alternative mechanisms. However, how this initiation is occurring and regulated remains unknown. Thus, to understand bacterial physiology and antibiotic resistance, we need to reveal the molecular mechanism of leaderless translation. This proposal aims to address this need. We will use single-molecule methods that are particularly well suited for the dissection of dynamic processes, such as translation. We developed a single-molecule fluorescence system that allows us to observe the entire initiation process. We can directly follow mRNA, ribosomal subunits, and translation factors, thus defining how initiation is occurring and regulated. In aim 1, we will determine how leaderless mRNAs are recruited to the ribosomes and how the start codon is recognized. We will directly test for the existence of the proposed alternative initiation mechanisms. Namely, we will define if mRNAs are directly recruited to the 70S ribosomes. We will then determine if start codon recognition is concurrent with mRNA recruitment, as was previously proposed. We will also determine the role of initiation factors in regulation of leaderless translation. In Aim 2, we will focus on the roles of mRNA sequence and structure. Translation efficiency of leaderless mRNAs is regulated by the start codon sequence, presence of nucleotides upstream of the start codon, and mRNA secondary structure. We will use our single-molecule toolkit to determine how these elements regulate translation. In particular, we are interested in how they affect alternative initiation pathways.

概括 无领导者 mRNA 缺少 5' UTR 和 SD 序列。此类 mRNA 在细菌和古细菌中很常见， 分枝杆菌是最引人注目的例子，因为大约三分之一的分枝杆菌 mRNA 是天然存在的。 群龙无首。无领导翻译是细菌应激反应和适应机制的一部分。在 与众所周知的含有 Shine Dalgarno 的 mRNA 相比，我们对起始过程知之甚少 无领导者 mRNA 的机制。无前导 mRNA 的生化、实验和系统分析 表达表明无领导者 mRNA 通过替代机制进行翻译和调节。 然而，这种启动是如何发生和调节的仍然未知。因此，要了解细菌 生理学和抗生素耐药性，我们需要揭示无领导翻译的分子机制。 本提案旨在解决这一需求。我们将使用特别适合的单分子方法 用于剖析动态过程，例如翻译。我们开发了单分子荧光 系统使我们能够观察整个启动过程。我们可以直接追踪mRNA、核糖体 亚基和翻译因子，从而定义启动是如何发生和调节的。在目标 1 中，我们将 确定无前导序列 mRNA 如何被招募到核糖体以及如何识别起始密码子。我们 将直接测试所提议的替代启动机制是否存在。也就是说，我们将定义如果 mRNA 直接募集至 70S 核糖体。然后我们将确定起始密码子识别是否是 正如之前提出的，与 mRNA 招募同时发生。我们还将确定启动的角色 无领导翻译的监管因素。在目标 2 中，我们将重点关注 mRNA 序列和 结构。无前导序列 mRNA 的翻译效率受起始密码子序列、存在 起始密码子上游的核苷酸和 mRNA 二级结构。我们将使用我们的单分子 确定这些元素如何调节翻译的工具包。我们特别感兴趣的是它们如何影响 替代起始途径。