Division of Pharmaceutical Sciences, Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA.
Department of Molecular Microbiology, Center for Advanced Laboratory Medicine, University of California, San Diego, La Jolla, CA, USA.
Nat Chem Biol. 2021 Aug;17(8):906-914. doi: 10.1038/s41589-021-00817-3. Epub 2021 Jun 17.
The development of unnatural base pairs (UBPs) has greatly increased the information storage capacity of DNA, allowing for transcription of unnatural RNA by the heterologously expressed T7 RNA polymerase (RNAP) in Escherichia coli. However, little is known about how UBPs are transcribed by cellular RNA polymerases. Here, we investigated how synthetic unnatural nucleotides, NaM and TPT3, are recognized by eukaryotic RNA polymerase II (Pol II) and found that Pol II is able to selectively recognize UBPs with high fidelity when dTPT3 is in the template strand and rNaMTP acts as the nucleotide substrate. Our structural analysis and molecular dynamics simulation provide structural insights into transcriptional processing of UBPs in a stepwise manner. Intriguingly, we identified a novel 3'-RNA binding site after rNaM addition, termed the swing state. These results may pave the way for future studies in the design of transcription and translation strategies in higher organisms with expanded genetic codes.
非天然碱基对（UBPs）的发展极大地提高了 DNA 的信息存储容量，使得通过异源表达的 T7 RNA 聚合酶（RNAP）在大肠杆菌中转录非天然 RNA 成为可能。然而，对于细胞 RNA 聚合酶如何转录 UBPs，人们知之甚少。在这里，我们研究了合成的非天然核苷酸 NaM 和 TPT3 如何被真核 RNA 聚合酶 II（Pol II）识别，并发现当模板链上存在 dTPT3 时，Pol II 能够以高保真度选择性地识别 UBPs，而 rNaMTP 则作为核苷酸底物。我们的结构分析和分子动力学模拟提供了一步一步转录加工 UBPs 的结构见解。有趣的是，我们在 rNaM 添加后鉴定出了一个新的 3'-RNA 结合位点，称为摆动态。这些结果可能为未来在高等生物中设计扩展遗传密码的转录和翻译策略铺平道路。
