Mechanism of Translation Initiation on Leaderless mRNAs

摘要 无前导序列的mRNAs缺少5‘非编码区和SD序列。这种mRNAs在细菌和古生菌中很常见， 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mRNAs是天然的 无人领导。无领导翻译是细菌应激反应和适应机制的一部分。在……里面 与众所周知的含有Shine Dalgarno的mRNAs相比，人们对其启动过程知之甚少 无领导的mRNAs的机制。无铅信使核糖核酸的生化、实验和系统分析 表达表明，无领导的mRNAs是通过其他机制翻译和调节的。 然而，这种启动是如何发生和调控的仍不清楚。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。 这项提议旨在满足这一需求。我们将使用特别适合的单分子方法 用于动态过程的剖析，例如翻译。我们开发了一种单分子荧光 使我们能够观察整个启动过程的系统。我们可以直接跟随mRNA，核糖体 亚单位和翻译因素，从而定义启动是如何发生和调节的。在目标1中，我们将 确定如何将无领导的mRNAs招募到核糖体中，以及如何识别起始密码子。我们 将直接测试拟议的替代启动机制的存在。也就是说，我们将定义是否 MRNA被直接招募到70年代的核糖体中。然后我们将确定起始密码子识别是否 与先前提出的信使核糖核酸招募同时进行。我们还将确定启蒙的作用 影响无领导翻译规范的因素。在目标2中，我们将重点关注mRNA序列和 结构。无前导mRNAs的翻译效率受起始密码子序列、 起始密码子上游的核苷酸和信使核糖核酸二级结构。我们将使用我们的单分子 确定这些元素如何规范翻译的工具包。特别是，我们感兴趣的是它们如何影响 另一种启动途径。

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.