State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Int J Mol Sci. 2023 Mar 3;24(5):4934. doi: 10.3390/ijms24054934.
T7 RNA polymerase is the most widely used enzyme in RNA synthesis, and it is also used for RNA labeling in position-selective labeling of RNA (PLOR). PLOR is a liquid-solid hybrid phase method that has been developed to introduce labels to specific positions of RNA. Here, we applied PLOR as a single-round transcription method to quantify the terminated and read-through products in transcription for the first time. Various factors, including pausing strategies, Mg, ligand and the NTP concentration at the transcriptional termination of adenine riboswitch RNA have been characterized. This helps to understand transcription termination, which is one of the least understood processes in transcription. Additionally, our strategy can potentially be used to study the co-transcription behavior of general RNA, especially when continuous transcription is not desired.
T7 RNA 聚合酶是最广泛应用于 RNA 合成的酶,也用于 RNA 标记的位置选择性 RNA 标记(PLOR)。PLOR 是一种液-固混合相方法,已被开发用于将标记物引入 RNA 的特定位置。在这里,我们首次将 PLOR 作为一种单轮转录方法应用于定量分析转录中的终止和通读产物。各种因素,包括暂停策略、Mg、配体和腺嘌呤核糖体开关 RNA 转录终止时的 NTP 浓度,都已经被表征。这有助于理解转录终止,这是转录中最不为人知的过程之一。此外,我们的策略可能被用于研究一般 RNA 的共转录行为,特别是当不希望连续转录时。