编码区的无义突变将氨基酸密码子转变为终止密码子,导致过早终止密码子(PTC)。在框架内PTC的情况下,如果翻译不停止在PTC,但继续插入氨基酸的自然终止密码子(NTC),被称为readthrough,形成全长肽,尽管只有一个氨基酸突变。我们以前开发了功能转移寡核苷酸(FT-探针),与互补序列的RNA形成杂合复合物以转移官能团,导致胞嘧啶的4-氨基或腺嘌呤的6-氨基的修饰。在这项研究中,FT-探针用于化学修饰PTC的腺苷(UAA,UAG,和UGA)的mRNA,在重组的大肠杆菌翻译系统中对其进行了读取分析。第三个腺苷修饰的UAA产生了三个掺入酪氨酸的连读肽,UAA位点的谷氨酰胺和赖氨酸。应当注意的是,用环糊精的额外修饰仅诱导谷氨酰胺掺入。腺苷修饰的UGA通过选择性色氨酸掺入非常有效地诱导读通。修饰的UGA的读透是由RF2功能的抑制引起的。这项研究表明,PTC的腺苷6-氨基的化学修饰是在原核翻译系统中有效读取的策略。
Nonsense mutations in the coding region turn amino acid codons into termination codons, resulting in premature termination codons (PTCs). In the case of the in-frame PTC, if translation does not stop at the PTC but continues to the natural termination codon (NTC) with the insertion of an amino acid, known as
readthrough, the full-length peptide is formed, albeit with a single amino acid mutation. We have previously developed the functionality-transfer oligonucleotide (FT-Probe), which forms a hybrid complex with RNA of a complementary sequence to transfer the functional group, resulting in modification of the 4-amino group of cytosine or the 6-amino group of adenine. In this study, the FT-Probe was used to chemically modify the adenosines of the PTC (UAA, UAG, and UGA) of mRNA, which were assayed for the
readthrough in a reconstituted Escherichia coli translation system. The third adenosine-modified UAA produced three
readthrough peptides incorporating tyrosine, glutamine and lysine at the UAA site. It should be noted that the additional modification with a cyclodextrin only induced glutamine incorporation. The adenosine modified UGA induced
readthrough very efficiently with selective tryptophan incorporation.
Readthrough of the modified UGA is caused by inhibition of the RF2 function. This study has demonstrated that the chemical modification of the adenosine 6-amino group of the PTC is a strategy for effective readthrough in a prokaryotic translation system.