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

描述(申请人提供)：生物体的生长和生存依赖于将其基因组信息忠实和快速地解码成功能性多肽序列。高精度的蛋白质合成确保了错误的多肽不会产生，这些多肽更容易错误折叠，因此可能会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，核糖体是负责将信使RNA解码为生命所有领域中的蛋白质的两个亚单位的大分子机器。在每个伸长周期中，核糖体从大量相互竞争的AA-tRNA中谨慎地选择与解码中心密码子匹配的适当的氨基酰基-tRNA(AA-tRNA)。除此之外，我们最近还发现了多肽键形成后核糖体上的质量控制机制，这有助于高保真蛋白质的合成。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已经到位，可以监测刚刚完成的化学步骤的质量。在延伸周期中，错误的氨基酸的加入被发现对后续反应的特异性有显著影响。这种反复积累的错误导致释放因子导致蛋白质合成的流产终止，在正常情况下，释放因子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背后的分子机制。我们的直接目标是找出信号是如何从干扰的mRNA-tRNA相互作用传递到解码中心的，这最终导致低保真蛋白质合成。我们还感兴趣的是，在干扰的mRNA-tRNA相互作用存在的情况下，释放因子的活性是如何调节在正义密码子上的，以及导致这一活性的结构线索。这些目标是在突变的翻译成分和低分辨率结构探测技术的背景下，围绕稳定前的动力学方法而建立的。作为第三个目标，我们感兴趣的是探索释放因子3在质量控制机制中以前未知的作用及其在细胞存活中的作用。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，并确定在这个过程中可能涉及的其他因素。 与公共卫生相关：重组蛋白质技术处于生产许多治疗剂的过程的前沿。从这里提出的研究中获得的信息对目前进行这一过程的方法有直接的影响，特别是过度表达的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标，拟议的研究可能会阐明它们的作用模式，以便做出更有效的治疗方法。

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.