The molecular mechanism of post-peptidyl quality control on the ribosome

描述（由申请人提供）：生物体的生长和活力取决于其基因组信息忠实和快速解码为功能肽序列。高精度的蛋白质合成确保不会产生更容易错误折叠并因此可能具有不期望的毒性后果的错误多肽。蛋白质合成的整体保真度似乎受到核糖体作用的限制，核糖体是负责在生命的所有领域将信使RNA解码为蛋白质的双亚基大分子机器。在每个延伸周期中，核糖体从大量相互竞争的氨酰tRNA中仔细选择与解码中心密码子相匹配的合适的氨酰tRNA。除此之外，我们最近还发现了一种发生在肽键形成后的核糖体质量控制机制，这有助于高保真蛋白质合成。类似于DNA和RNA聚合酶以及tRNA合成酶所享有的校对策略，新发现的基于核糖体的机制已经到位，可以监控刚刚完成的化学步骤的质量。在延伸循环期间，发现掺入不正确的氨基酸对随后反应的特异性具有显著影响。这种错误的反复积累导致蛋白质合成被释放因子终止，而释放因子在正常条件下很少解码有义密码子。我们工作的长期目标是彻底了解这一过程的分子机制。我们的近期目标是找出信号是如何从受干扰的mRNA-tRNA相互作用传递到解码中心的，这最终导致了低保真度的蛋白质合成。我们还感兴趣的释放因子的活动是如何在有义密码子上的扰动mRNA- tRNA相互作用的存在下进行调节，以及负责这种活动的结构线索。这些目标是建立在突变的翻译组件的背景下，和低分辨率的结构探测技术的前稳态动力学方法。作为第三个目标，我们感兴趣的是探索释放因子3在质量控制机制及其在细胞活力中的效用中的先前未知的作用。最后，我们感兴趣的是发现这个系统是否存在于真核生物中，并确定其他因素，如果有的话，可能涉及在这个过程中。 公共卫生相关性：重组蛋白技术是许多治疗药物生产过程的最前沿。从本文提出的研究中获得的信息对目前进行该过程的方法具有直接影响，特别是过度表达蛋白质的质量和产量似乎密切相关。此外，由于核糖体是许多抗生素的靶点，因此拟议的研究可能会揭示它们的作用模式，以便制定更有效的治疗方法。

DESCRIPTION (provided by applicant): Organismal growth and viability is dependent on the faithful and fast decoding of its genomic information into functional peptide sequences. High-accuracy protein synthesis ensures that errant polypeptides, which are more prone to misfold and hence may have undesirable toxic consequences, are not produced. The overall fidelity of protein synthesis appears to be limited by the action of the ribosome, which is the two-subunit macromolecular machine responsible for the decoding of the messenger RNA into protein in all domains of life. During each cycle of elongation, the ribosome carefully selects the appropriate aminoacyl-tRNA (aa-tRNA) that matches the codon in the decoding center from a large-pool of competing aa-tRNAs. In addition to this, we have recently uncovered a quality control mechanism on the ribosome that takes place after peptide-bond formation, which contributes to high-fidelity protein synthesis. Akin to the proofreading strategies enjoyed by DNA and RNA polymerases and tRNA synthetases, the newly discovered ribosome-based mechanism is in place to monitor the quality of the just completed chemical step. During the elongation cycle, the incorporation of an incorrect amino acid was found to have dramatic effects on the specificity of the subsequent reaction. This iterated accumulation of errors results in the abortive termination of protein synthesis by release factors, which under normal conditions rarely decode sense codons. The long term goal of our work is to gain a thorough understanding of the molecular mechanisms underlying this process. Our immediate goal is to find out how the signal is communicated from a perturbed mRNA-tRNA interaction to the decoding center, which ultimately leads to low-fidelity protein synthesis. We are also interested in how the activity of release factors is modulated on sense codons in the presence of a perturbed mRNA- tRNA interaction, and the structural cues that are responsible for this activity. These goals are built around pre-steady state kinetics approaches in the context of mutated translation components, and low-resolution structural probing techniques. As a third goal we are interested in exploring a previously unknown role for release factor 3 in the quality control mechanism and its utility in cellular viability. Finally we are interested in finding whether this system exists in eukaryotes, and identifying other factors, if any, that might be involved during this process. PUBLIC HEALTH RELEVANCE: Recombinant protein technologies are at the forefront of the process by which many therapeutic agents are produced. Information obtained from the research proposed here has immediate ramifications for the means by which this process is currently carried out, especially that the quality and yield of over- expressed proteins appear to be intimately correlated. Furthermore, as the ribosome is the target of many antibiotics, the proposed research is likely to shed light into their mode of action in order to make more effective therapeutics.