Mechanism of Translation Initiation on Leaderless mRNAs
无领导者 mRNA 的翻译起始机制
基本信息
- 批准号:10593807
- 负责人:
- 金额:$ 22.17万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:5&apos Untranslated RegionsAddressAffectAntibiotic ResistanceArchaeaBacteriaBacterial PhysiologyBase PairingBindingBiochemicalCellsCodon NucleotidesDissectionElementsEukaryotaFluorescenceFluorescence Resonance Energy TransferGenus MycobacteriumGuanosine TriphosphateHydrolysisInitiator CodonInitiator tRNALabelMessenger RNAMethodsMicroscopyModelingMolecularMolecular ConformationNatureNucleotidesPaintPathway interactionsPeptide Initiation FactorsPlayPrevalenceProcessProtein BiosynthesisProteinsProteomeRegulationRibosomal RNARibosomesRoleStructureSystemTestingTextbooksTimeTransfer RNATranslatingTranslation InitiationTranslationsanalogbiological adaptation to stressin vivointerestmRNA ExpressionmRNA Translationmycobacterialnegative affectnovelrecruitsingle moleculetranslation factor
项目摘要
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.
总结
无前导序列的mRNA缺乏5'UTR和SD序列。这种mRNA在细菌和细菌中很常见,
分枝杆菌是最突出的例子,因为大约三分之一的分枝杆菌mRNA天然地
没有领导无领导翻译是细菌应激反应和适应机制的一部分。在
与已知的含Shine Dalgarno的mRNA相比,
leaderless mRNAs的机制。无前导基因mRNA的生物化学、实验和系统分析
表达表明,无前导mRNA的翻译和调节通过替代机制。
然而,这种启动是如何发生和调节的仍然是未知的。因此,为了了解细菌
生理学和抗生素抗性,我们需要揭示无前导翻译的分子机制。
本提案旨在满足这一需要。我们将使用单分子方法,
对于动态过程的剖析,例如翻译。我们开发了一种单分子荧光
这个系统可以让我们观察整个启动过程。我们可以直接跟踪mRNA,核糖体
亚基和翻译因子,从而定义了起始如何发生和调节。在目标1中,
确定无前导mRNA如何被募集到核糖体以及起始密码子如何被识别。我们
将直接测试所提议的替代启动机制的存在。也就是说,我们将定义,如果
mRNA被直接募集到70S核糖体。然后我们将确定起始密码子识别是否是
与先前提出的mRNA募集同时进行。我们还将确定启蒙的作用
无领导翻译的调控因素。在目标2中,我们将重点关注mRNA序列的作用,
结构无前导基因mRNA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mRNA二级结构。我们将用我们的单分子
工具包来确定这些元素如何调节翻译。特别是,我们感兴趣的是它们如何影响
替代启动途径。
项目成果
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