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

描述（由申请人提供）：生物体的生长和生存能力取决于其基因组信息的忠实和快速解码成功能肽序列。高精度的蛋白质合成确保不会产生错误的多肽，这些多肽更容易发生错误折叠，从而可能产生不良的毒性后果。蛋白质合成的整体保真度似乎受到核糖体作用的限制，核糖体是一种由两个亚基组成的大分子机器，负责在生命的所有领域将信使RNA解码成蛋白质。在每个延伸周期中，核糖体从大量竞争的aa-tRNA中仔细选择与解码中心密码子匹配的氨基酸基trna （aa-tRNA）。除此之外，我们最近发现了发生在肽键形成后的核糖体上的质量控制机制，这有助于高保真蛋白的合成。类似于DNA和RNA聚合酶以及tRNA合成酶的校对策略，新发现的基于核糖体的机制可以监测刚刚完成的化学步骤的质量。在延伸周期中，发现不正确的氨基酸的掺入对随后反应的特异性有显著影响。这种错误的反复积累导致释放因子终止蛋白质合成，而释放因子在正常条件下很少解码义密码子。我们工作的长期目标是彻底了解这一过程背后的分子机制。我们的直接目标是找出信号如何从受干扰的mRNA-tRNA相互作用传递到解码中心，最终导致低保真蛋白合成。我们也对在mRNA- tRNA相互作用受到干扰的情况下释放因子的活性是如何在感觉密码子上被调节的，以及负责这种活性的结构线索感兴趣。这些目标是围绕在突变翻译成分和低分辨率结构探测技术背景下的预稳态动力学方法建立的。作为第三个目标，我们感兴趣的是探索释放因子3在质量控制机制中的未知作用及其在细胞活力中的效用。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，并确定在这个过程中可能涉及的其他因素，如果有的话。

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.