Mechanisms regulating speed and accuracy of translation

蛋白质合成的速度和准确性对细胞的生存和适应不断变化的环境条件至关重要。在翻译过程中，快速合成的时期被停顿所打断；人们对这些停顿的具体性质和持续时间知之甚少。在这个项目中，我们建议通过结合时间分辨的高性能体外翻译分析和随机建模来分析翻译停顿。一个特殊的情况是翻译暂停当串脯氨酸残基被纳入。这种失速可以通过伸长因子P （EF-P）来缓解。我们将研究为什么脯氨酸片段会抑制核糖体的肽基转移酶活性，以及EF-P如何通过使用脯氨酸类似物工具箱测量富含脯氨酸序列的合成速率，并将类似物的化学性质与翻译效率进行比较，来拯救核糖体。mRNA特定区域的翻译延迟可能促进共翻译折叠。我们将跟踪新生肽的合成，通过核糖体出口通道的共翻译运动，并通过时间分辨FRET （Förster共振能量转移）和PET（光诱导电子转移）方法使用非规范氨基酸衍生物的共翻译插入来折叠它们。配合物在隧道运动和折叠过程中的结构将通过低温电子显微镜来解决，并通过分子动力学模拟来模拟。为了了解蛋白质合成的速度和准确性之间的平衡，我们计划使用质谱法系统地评估细菌体内蛋白质合成的误差频率。我们将确定哪些错误是普遍存在的，细胞如何对亚致死剂量的错误诱导抗生素作出反应，以及蛋白质组如何对高错误负荷作出反应。两个翻译gtpase （SelB和IF2）的功能和结构将被研究。该项目将展示蛋白质合成的速度和准确性如何影响细胞蛋白质组的质量。

Speed and accuracy of protein synthesis are crucial for the survival of cells and their adaptation to changing environmental conditions. During ongoing translation, periods of rapid synthesis are interrupted by pauses; the detailed nature and duration of those pauses remains poorly understood. In this project we propose to analyze translation pauses by a combination of time-resolved high-performance in-vitro translation assays and stochastic modeling. One special case is translation pausing when strings of proline residues are to be incorporated. This stalling is alleviated by elongation factor P (EF-P). We will study why stretches of prolines inhibit the peptidyl transferase activity of the ribosome and how EF-P rescues the ribosome from stalling by measuring the rates of synthesis of proline-rich sequences using a toolbox of proline analogs and comparing the chemical properties of the analogs with the efficiency of translation. Translation delays in specific regions of the mRNA may facilitate co-translational folding. We will follow the synthesis, co-translational movement of nascent peptides through the exit tunnel of the ribosome, and their folding by time-resolved FRET (Förster Resonance Energy Transfer) and PET (Photoinduced Electron Transfer) methods using co-translational insertion of non-canonical amino acid derivatives. The structure of the complexes during movement through the tunnel and folding will be solved by cryo-EM and modelled by molecular dynamic simulations.To understand the balance between the speed and accuracy of protein synthesis, we plan to systematically evaluate the error frequencies of protein synthesis in vivo in bacteria using mass spectrometry. We will determine which errors are prevalent, how cells respond to sub-lethal doses of error-inducing antibiotics and how the proteome responds to high error loads. Function and structure of two translational GTPases (SelB and IF2) will be studied. The project will show how the speed and accuracy of protein synthesis contribute to the quality of the cellular proteome.