Structural bases for cellular stress responses mediated by stalled translation

翻译停滞介导的细胞应激反应的结构基础

• 批准号: 8595445
• 负责人: Andrei Korostelev
• 金额: $ 31.64万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8595445
• 关键词: Address; Adopted; Affinity; Amino Acids; Amino Acyl Transfer RNA; Aminoacyl-tRNA hydrolase; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Bacterial Proteins; Binding; Binding Sites; Biochemical; C-terminal; Cause of Death; Cells; Cellular Stress Response; Codon Nucleotides; Collaborations; Complex; Crystallization; Data; Development; Enzymes; Exposure to; Free Ribosome; GTP Pyrophosphokinase; Gene Expression; Hydrolysis; Image; Immune response; Infection; Kinetics; Lead; Letters; Mediating; Messenger RNA; Methods; Modeling; Modification; Molecular; Molecular Conformation; N-terminal; Nutrient; Nutritional; Pathogenicity; Pathway interactions; Peptides; Play; Process; Protein Biosynthesis; Proteins; Recycling; Regulation; Research; Resources; Ribosomes; Role; Sense Codon; Site; Solubility; Starvation; Stress; Structure; Testing; Time; Transfer RNA; Translating; Translation Initiation; Translations; Ursidae Family; Virulence; Work; base; biological adaptation to stress; deprivation; design; mRNA Transcript Degradation; next generation; nutrition; peptidyl-tRNA; premature; programs; public health relevance; release factor; response; single molecule; small molecule; tmRNA; translation factor

DESCRIPTION (provided by applicant): Bacterial pathogenicity and ability to survive in adverse conditions depends on bacterial stress response. This proposal aims at a structural understanding of two bacterial stress- response processes, in which stalled non-translating ribosomes are being sensed: 1) stringent response, which is mediated by stringent factor RelA; and 2) rescue of stalled ribosomes by a peptidyl- tRNA hydrolase YaeJ. Bacteria adapt to insufficient nutritional conditions via a mechanism termed the stringent response. One of the consequences of nutrient deprivation is amino acid starvation, which may lead to more than a 5-fold increase in cellular levels of uncharged (deacylated) tRNAs. Deacylated tRNAs cannot participate in protein synthesis but can bind to ribosomes, which are in a paused translational state due to insufficient levels of aminoacylated tRNAs. Such stalled ribosomes are thought to interact with RelA and initiate the stringent response. RelA is an 84 kDa enzyme, which, upon binding to stalled ribosomes, catalyzes the synthesis of the small molecule "alarmones" ppGpp and pppGpp. These molecules trigger the stringent response by initiating a global gene expression program. The molecular mechanism of the RelA-mediated stringent response is poorly understood. First, the binding site for RelA on the ribosome has not been identified. Second, it is not known how the presence of deacylated tRNAs on the ribosome triggers the (p)ppGpp-synthesizing activity of RelA. In Specific Aim 1, we propose to address these questions by obtaining structural and dynamics information on 70S*RelA ribosome complexes. In addition to nutrient-deprivation conditions, other cellular conditions exist that result in mRNA degradation or modification, interfere with aminoacyl-tRNA binding to the A site, tRNA translocation or other steps of translation elongation. This leads to the stalling of translating ribosomes. In this stalled state, peptidyl-tRNA is stably bound to the ribosomal P site, and the ribosome is not available for initiation of translation on a new mRNA. Because ribosome synthesis requires large amounts of cell resources, it is essential that non-translating ribosomes be recycled and not degraded. To rescue such ribosomes, the incomplete protein chains and tRNAs have to be released from the ribosomes. At least two mechanisms exist, namely the well-characterized tmRNA-assisted ribosome rescue and a recently proposed YaeJ-mediated peptide release. YaeJ is a 16 kDa protein that is hypothesized to directly catalyze peptidyl-tRNA hydrolysis on the ribosome in a codon-independent manner. Our Specific Aim 2 is designed to address mechanistic questions concerning YaeJ-mediated response to ribosome stalling. The proposed aims will be accomplished by structural and biochemical methods.

描述（由申请人提供）：细菌的致病性和在不利条件下生存的能力取决于细菌的应激反应。该提案旨在从结构上理解两种细菌应激反应过程，其中停滞的非翻译核糖体被感知：1）严格反应，由严格因子 RelA 介导； 2) 通过肽基-tRNA 水解酶 YaeJ 拯救停滞的核糖体。细菌通过一种称为严格反应的机制来适应营养不足的条件。营养缺乏的后果之一是氨基酸饥饿，这可能导致细胞中不带电（脱酰基）tRNA 的水平增加 5 倍以上。脱酰化 tRNA 不能参与蛋白质合成，但可以与核糖体结合，由于氨酰化 tRNA 水平不足，核糖体处于暂停翻译状态。这种停滞的核糖体被认为与 RelA 相互作用并引发严格的反应。 RelA 是一种 84 kDa 的酶，在与停滞核糖体结合后，催化小分子“警报素”ppGpp 和 pppGpp 的合成。这些分子通过启动全局基因表达程序来触发严格的反应。人们对 RelA 介导的严格反应的分子机制知之甚少。首先，RelA 在核糖体上的结合位点尚未确定。其次，尚不清楚核糖体上脱酰化 tRNA 的存在如何触发 RelA 的 (p)ppGpp 合成活性。在具体目标 1 中，我们建议通过获取 70S*RelA 核糖体复合物的结构和动力学信息来解决这些问题。除了营养缺乏条件外，还存在其他导致 mRNA 产生的细胞条件 降解或修饰，干扰氨酰基-tRNA 与 A 位点的结合、tRNA 易位或翻译延伸的其他步骤。这导致核糖体翻译停滞。在这种停滞状态下，肽基-tRNA 稳定地结合到核糖体 P 位点，并且核糖体无法启动新 mRNA 的翻译。由于核糖体合成需要大量的细胞资源，因此非翻译核糖体的回收和不降解至关重要。为了拯救这种核糖体，必须从核糖体中释放不完整的蛋白质链和 tRNA。至少存在两种​​机制，即充分表征的 tmRNA 辅助核糖体救援和最近提出的 YaeJ 介导的肽释放。 YaeJ 是一种 16 kDa 的蛋白质，假设它以不依赖于密码子的方式直接催化核糖体上的肽基-tRNA 水解。我们的具体目标 2 旨在解决有关 YaeJ 介导的核糖体停滞反应的机制问题。拟议的目标将通过结构和生化方法来实现。