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

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），寡聚脯氨酸片段与不同的类似物有不同的翻译。与Rodnina和Wilson的团队合作，我们使用了一系列脯氨酸类似物，以了解延伸因子P（EF-P）在寡脯氨酸片段翻译中的机制。使用一组描述符（例如侧链立体，诱导效应，pKa），使我们能够确定脯氨酸puckering作为影响相对翻译速率的主导因素。我们进一步在用正交对表征翻译方面取得了进展，并发现通常观察到的正交翻译的低效率不仅仅是由于正交tRNA和释放因子（RF）之间的竞争，而是由于核糖体停滞。我们开始全面阐明EF的机制-在蛋白质合成中使用化学上不同的脯氨酸类似物的靶序列和分子动力学模拟。首先，我们将生成一个化学肽模型来测量各种类似物的酰胺键形成速率，并将这些速率与核糖体速率进行比较。这将在与P4和P6集团业已建立的合作框架内进行。此外，我们将进行广泛的核糖体EF-P复合物的分子动力学模拟，以进一步研究聚脯氨酸延伸诱导细菌核糖体失速的机制，以及这种失速是如何通过EF-P解决的。我们将继续我们的研究，旨在了解失速在翻译中的作用，与P6（参数化）和P7（分析）合作的正交对。我们计划在有或没有RF 1的情况下精确定位细胞mRNA库上停滞的核糖体的位置。在存在不同数量的框内琥珀终止密码子的情况下，将通过我们的报告基因活性和定量测定法对正交对效率进行进一步表征。此外，我们将使用吡咯赖氨酸（Pyl）为基础的o-对（PylRS：tRNAPyl）结合不同的修饰类似物，以阐明的作用，在EF-P的crosslysyl-Lys 34修饰。结合交联类似物和优化的正交翻译设置，从我们的工作将提供新的，有效的工具，为未来的结构以及共翻译折叠研究。

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.