Genetic encoding of Post-translational modifications

翻译后修饰(PTM)是细胞内核糖体蛋白合成后，特定酶作用于蛋白质侧链或末端的共价修饰。PTMS在多种生物过程中起着重要的调节作用，是自然界用来增加蛋白质中化学官能团的方法之一。精氨酸甲基化是一种重要的PTM，它调节许多蛋白质的活性，参与RNA加工、转录调控、信号转导和DNA修复。酪氨酸磷酸化，另一种PTM，在细胞周期进程、细胞增殖、细胞动态平衡、神经活动、转录激活和衰老中发挥重要作用。许多疾病，包括癌症和神经疾病，都与PTMS水平异常有关。尽管PTMS在生物学上很重要，但通常很难获得具有特定PTMS的同质蛋白质样本。这主要是因为细胞有相互竞争的机制来安装和移除PTM，而且翻译后修饰的蛋白质样本也含有未修饰的蛋白质。通常很难将翻译后修饰的蛋白质与其未修饰的类似物分开。如果蛋白质有多个PTM位点，问题就会进一步复杂化。此外，蛋白质组学分析揭示了许多蛋白质上的许多以前未确定的PTM位点。通常情况下，安装这些PTM的酶是未知的，因此进一步限制了对这些翻译后修饰蛋白的访问。为了应对这些挑战，我们建议开发基因定点结合甲基精氨酸和磷酸酪氨酸的方法。这项工作将为获得几种重要的翻译后修饰蛋白质提供便利的途径。蛋白质是由一种叫做核糖体的生物机器在所有生物体中合成的。核糖体使用信使RNA(MRNA)作为模板，转移RNA(TRNA)作为适配分子，利用遗传密码定义的一系列规则催化蛋白质合成。对于每一种天然存在的氨基酸，1)DNA或mRNA中有一组独特的三联体碱基，称为密码子，与氨基酸相对应，以及2)一组独特的氨基酰基-tRNA合成酶(AARs)/tRNA对。AARS催化其相应的tRNAs与其相应的氨基酸充电。在蛋白质合成过程中，带电的tRNA被带到核糖体，在那里它与核糖体小亚基中的mRNA上的同源密码子结合，同时在大亚基中的生长肽链中添加一个氨基酸。通过将琥珀终止密码子或四重密码子分配给非天然氨基酸，并向细胞提供正交的Aars/tRNA对，实现了非天然氨基酸在细胞中的遗传掺入。正交的AARs/tRNA对不干扰宿主细胞的tRNA合成酶和tRNA，并特异性地识别非天然氨基酸。使用这种方法，许多非天然氨基酸在不同的生物体中被遗传编码。然而，只有一小部分PTM是通过基因编码的。在这项建议中，我们探索了不同的方法来在大肠杆菌中识别新的正交AARS/tRNA对，以实现甲基精氨酸和磷酸酪氨酸的遗传编码。

Post-translational modifications (PTMs) are covalent modifications on side chains or termini of proteins installed by specific enzymes after ribosomal protein synthesis in cells. PTMs play an important role in modulating several biological processes and is one of the methods used by nature to increase the repertoire of chemical functional groups in proteins. Methylation of arginine is an important PTM that modulates the activity of numerous proteins involved in RNA processing, transcriptional regulation, signal transduction and DNA repair. Tyrosine phosphorylation, another PTM, that plays an important role in cell-cycle progression, cell proliferation, cellular homeostasis, neural activity, transcriptional activation and ageing. Many diseases, including cancer and neurological disorders, are associated with abnormal levels of PTMs. Despite the importance of PTMs in biology, it is often difficult to obtain a homogeneous sample of protein with specific PTMs. This is mainly because cells have competing mechanisms to install and remove PTMs, and samples of post-translationally modified proteins also contain unmodified proteins. It is often difficult to separate post-translationally modified proteins from their unmodified analogues. The problem is further compounded if the protein has multiple sites for PTMs. In addition, proteomics analyses have revealed numerous previously unidentified sites of PTMs on many proteins. Often enzymes that install these PTMs are not known, thus further limiting the access to these post-translationally modified proteins. To address these challenges, here we propose to develop methods for genetic site-specific incorporation of methylarginines and phosphotyrosine. This work would provide facile access to several important post-translationally modified proteins. Proteins are synthesized in all organisms by a biological machine called the ribosome. The ribosome uses messenger RNA (mRNA) as a template and transfer RNA (tRNA) as an adaptor molecule to catalyse protein synthesis using a set of rules defined by the genetic code. For each naturally occurring amino acid there are, 1) a unique set of triplet bases in the DNA or mRNA, called codons, which correspond to the amino acid, and 2) a unique set of aminoacyl-tRNA synthetases (aaRS)/ tRNA pairs. aaRSs catalyze the charging of their corresponding tRNAs with their corresponding amino acids. During protein synthesis, the charged tRNA is taken to the ribosome where it binds to its cognate codon on the mRNA in the small subunit of the ribosome, and concurrently an amino acid is added to the growing peptide chain in the large subunit. Genetic incorporation of unnatural amino acids in cells has been achieved by assigning an amber stop codon or quadruplet codons to unnatural amino acids and supplying the cells with orthogonal aaRS/ tRNA pairs. Orthogonal aaRS/ tRNA pairs do not interfere with the tRNA synthetases and the tRNAs of the host cell, and specifically recognize the unnatural amino acid. Using this approach, numerous unnatural amino acids have been genetically encoded in different organisms. However, only a small number of PTMs have been genetically encoded. In this proposal, we explore different approaches to identify new orthogonal aaRS/ tRNA pairs in E. coli to enable genetic encoding of methylarginines and phosphotyrosine.