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生存的能力取决于细菌的应激反应。本研究旨在从结构上理解两种细菌应激反应过程，在这两种过程中，停滞的非翻译核糖体被检测到：1)严格反应，由严格因子RelA介导；2)肽基- tRNA水解酶YaeJ对停滞核糖体的拯救作用。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。营养剥夺的后果之一是氨基酸饥饿，这可能导致细胞中不带电（去酰化）trna水平增加5倍以上。去酰化的trna不能参与蛋白质合成，但可以与核糖体结合，由于氨基酰化的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相互作用并启动严格的反应。RelA是一种84 kDa的酶，与停滞的核糖体结合后，催化小分子“警报”ppGpp和pppGpp的合成。这些分子通过启动一个全球性的基因表达程序来触发严格的反应。rela介导的严格反应的分子机制尚不清楚。首先，RelA在核糖体上的结合位点尚未确定。其次，目前尚不清楚核糖体上脱酰基trna的存在如何触发RelA的(p) ppgpp合成活性。在Specific Aim 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。除了营养剥夺条件外，存在其他导致mRNA的细胞条件

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.