Structural bases for cellular stress responses mediated by stalled translation

描述(由申请人提供)：细菌的致病性和在不利条件下的生存能力取决于细菌的应激反应。这一建议旨在从结构上理解细菌的两个应激反应过程，在这两个过程中，停滞的非翻译核糖体被感觉到：1)严格的反应，这是由严格的因子relA介导的；以及2)通过肽基-tRNA水解酶YaeJ拯救停滞的核糖体。细菌通过一种被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，这可能导致细胞内未带电(脱酰)的tRNA水平增加5倍以上。脱酰化的tRNA不能参与蛋白质的合成，但可以与核糖体结合，核糖体由于氨基酰化的tRNA水平不足而处于暂停的翻译状态。这种停滞不前的核糖体被认为与relA相互作用，并启动严格的反应。RELA是一种84 kDa的酶，当与停滞的核糖体结合时，催化合成小分子“丙酮”ppGpp和pppGpp。这些分子通过启动全球基因表达程序来触发严格的反应。RelA介导的严谨反应的分子机制尚不清楚。首先，核糖体上的relA结合部位尚未确定。其次，目前尚不清楚核糖体上脱酰化的tRNAs如何触发RelA的(P)ppGpp合成活性。在具体目标1中，我们建议通过获得70年代*rela核糖体复合体的结构和动力学信息来解决这些问题。除了营养匮乏的条件外，还存在其他导致mrna的细胞条件。 降解或修饰，干扰氨酰-tRNA与A位点的结合、tRNA易位或其他步骤的翻译延长。这导致了核糖体翻译的停滞。在这种停滞状态下，多肽-tRNA稳定地结合到核糖体P位点，核糖体不能用于启动新的mRNA的翻译。由于核糖体的合成需要大量的细胞资源，因此必须对非翻译的核糖体进行循环利用，而不是降解。为了挽救这种核糖体，不完整的蛋白质链和tRNA必须从核糖体中释放出来。至少存在两种机制，即特征明确的tmRNA辅助的核糖体拯救和最近提出的YaeJ介导肽的释放。YaeJ是一种16 kDa的蛋白质，被认为是以不依赖于密码子的方式直接催化核糖体上的肽-tRNA水解酶。我们的具体目标2旨在解决与YaeJ介导的核糖体停滞反应有关的机制问题。拟议的目标将通过结构和生物化学方法来实现。

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.