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Probing the mechanisms of unnatural protein translation with an engineered genetic code and orthogonal translational pairs

利用工程遗传密码和正交翻译对探索非自然蛋白质翻译机制

基本信息

批准号：
220072616
负责人：
Professor Dr. Nediljko Budisa
金额：
--
依托单位：
Abteilung für theoretische und computergestützte Biophysik
依托单位国家：
德国
项目类别：
Research Units
财政年份：
2012
资助国家：
德国
起止时间：
2011-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/220072616?language=en
关键词：
Probing mechanisms unnatural protein translation

项目摘要

In our previous work we employed chemically distinct proline analogs to study translation of single prolines and oligo-proline stretches in target sequences. A key result of this work was the discovery (under in vivo expression conditions) that oligo-proline stretches are differently translated with different analogs. In collaboration with groups of Rodnina and Wilson we used a series of proline analogs in order to understand the mechanism of elongation factor P (EF-P) in translation of oligo-proline stretches. The use of a set of descriptors (e.g. side chain sterics, inductive effects, pKa), enabled us to identify proline puckering as a dominant factor affecting relative translational rates. We further made progress in characterizing translation with orthogonal pairs and found that the commonly observed inefficiency of orthogonal translation is not exclusively due to the competition between the orthogonal tRNA and release factors (RF) but rather due to ribosomal stalling.Based on these encouraging results, we set out to fully elucidate the mechanism of EF-P in protein synthesis by using chemically distinct proline analogs in target sequences and molecular dynamics simulations. First, we will generate a chemical peptide model to measure the rate of amide bond formation with various analogues and we will compare these rates with ribosomal rates. This will be carried out in the context of the already established cooperation with P4 and P6 groups. Furthermore, we will perform extensive molecular dynamics simulations of the ribosome EF-P complex to further investigate the mechanism by which poly-proline stretches induce stalling in bacterial ribosomes and how this stalling is resolved by EF-P.We will continue our studies aiming to understand the role of stalling in translation with orthogonal pairs in collaboration with P6 (parameterization) and P7 (profiling). We plan to pinpoint the location of stalled ribosomes on the cellular mRNA pool in presence or in absence of RF 1. Further characterization of orthogonal pair efficiency will be conducted via our reporter gene activity and quantification assays in the presence of a different number of in-frame amber stop codons. In addition, we will use pyrrolysine (Pyl)-based o-pairs (PylRS:tRNAPyl) combined with distinct modification analogs to elucidate the role of the ß lysyl-Lys34 modification in EF-P.Incorporation of crosslinking analogs and optimized orthogonal translation setups resulting from our work will provide new, efficient tools for future structural as well as co-translational folding studies.

在我们之前的工作中，我们采用化学上不同的脯氨酸类似物来研究靶序列中单个脯氨酸和寡聚脯氨酸片段的翻译。这项工作的一个关键结果是发现（在体内表达条件下）寡聚脯氨酸片段用不同的类似物进行不同的翻译。我们与 Rodnina 和 Wilson 小组合作，使用了一系列脯氨酸类似物来了解寡聚脯氨酸延伸翻译中延伸因子 P (EF-P) 的机制。使用一组描述符（例如侧链空间、诱导效应、pKa），使我们能够将脯氨酸褶皱确定为影响相对翻译速率的主要因素。我们进一步在用正交对表征翻译方面取得了进展，发现通常观察到的正交翻译效率低下并不完全是由于正交 tRNA 和释放因子 (RF) 之间的竞争，而是由于核糖体停滞。基于这些令人鼓舞的结果，我们着手通过在目标序列中使用化学上不同的脯氨酸类似物来充分阐明 EF-P 在蛋白质合成中的机制，以及分子动力学模拟。首先，我们将生成一个化学肽模型来测量与各种类似物形成酰胺键的速率，并将这些速率与核糖体速率进行比较。这将在与 P4 和 P6 集团已经建立的合作范围内进行。此外，我们将对核糖体 EF-P 复合物进行广泛的分子动力学模拟，以进一步研究聚脯氨酸拉伸诱导细菌核糖体停顿的机制以及 EF-P 如何解决这种停顿。我们将继续我们的研究，旨在与 P6（参数化）和 P7（分析）合作，了解正交对翻译中停顿的作用。我们计划在存在或不存在 RF 1 的情况下查明细胞 mRNA 池上停滞核糖体的位置。正交对效率的进一步表征将通过我们的报告基因活性和定量测定在存在不同数量的框内琥珀终止密码子的情况下进行。此外，我们将使用基于吡咯赖氨酸（Pyl）的o对（PylRS：tRNAPyl）与不同的修饰类似物相结合来阐明EF-P中β赖氨酰-Lys34修饰的作用。我们的工作产生的交联类似物和优化的正交翻译设置的结合将为未来的结构折叠和共翻译折叠提供新的、有效的工具研究。