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: 8858644
• 负责人: Andrei Korostelev
• 金额: $ 31.83万
• 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 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8858644
• 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; Health; 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; 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良条件下生存的能力取决于细菌应激反应。该建议旨在对两个细菌应激反应过程的结构理解，在这种过程中，正在感测下停滞的非翻译核糖体：1）严格的响应，这是由严格的因子Rela介导的；和2）肽基TRNA水解酶Yaej营救了失速的核糖体。细菌通过称为严格反应的机制适应营养条件不足。营养剥夺的后果之一是氨基酸饥饿，这可能会导致未充电（脱酰基化）TRNA的细胞水平增加5倍以上。脱酰化的TRNA不能参与蛋白质合成，但可以与核糖体结合，核糖体由于氨基化的TRNA水平不足而处于暂停的平移状态。这种停滞的核糖体被认为与RELA相互作用并启动严格的响应。 RERA是一种84 kDa酶，在与停滞的核糖体结合后，它催化了小分子“警报器” PPGPP和PPPGPP的合成。这些分子通过启动全球基因表达程序触发了严格的响应。相对介导的严格反应的分子机制知之甚少。首先，尚未鉴定出核糖体上Rela的结合位点。其次，尚不清楚核糖体上脱酰化的TRNA的存在如何触发RELA的（P）PPGPP合成活性。在特定的目标1中，我们建议通过在70S*Rela Ribosos络合物上获得结构和动态信息来解决这些问题。除了营养剥夺条件外，还有其他细胞条件会导致mRNA 降解或修饰，干扰与位点的氨基酰基-TRNA结合，tRNA易位或其他翻译伸长的步骤。这导致翻译核糖体的失速。在这种停滞状态下，肽基-TRNA稳定与核糖体P位点结合，核糖体不可用于在新的mRNA上启动翻译。由于核糖体合成需要大量的细胞资源，因此必须将非翻译核糖体回收并且不会降解至关重要。为了营救此类核糖体，必须从核糖体中释放出不完整的蛋白质链和TRNA。至少存在两种​​机制，即特征良好的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蛋白，假设以密码子独立的方式直接在核糖体上直接催化肽基-TRNA水解。我们的特定目标2旨在解决有关Yaej介导的对核糖体停滞的反应的机械问题。所提出的目标将通过结构和生化方法来实现。