Zhai Weiji, Duan Yanting, Zhang Xiaomei, Xu Guoqiang, Li Hui, Shi Jinsong, Xu Zhenghong, Zhang Xiaojuan
Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi, China.
Synth Syst Biotechnol. 2022 Jun 20;7(4):1046-1055. doi: 10.1016/j.synbio.2022.06.003. eCollection 2022 Dec.
The intrinsic terminator in prokaryotic forms secondary RNA structure and terminates the transcription. However, leaking transcription is common due to varied terminator strength. Besides of the representative hairpin and U-tract structure, detailed sequence and thermodynamic features of terminators were not completely clear, and the effect of terminator on the upstream gene expression was unclearly. Thus, it is still challenging to use terminator to control expression with higher precision. Here, in , we firstly determined the effect of the 3'-end sequences including spacer sequences and terminator sequences on the expression of upstream and downstream genes. Secondly, terminator mutation library was constructed, and the thermodynamic and sequence features differing in the termination efficiency were analyzed using the FlowSeq technique. The result showed that under the regulation of terminators, a negative correlation was presented between the expression of upstream and downstream genes (r=-0.60), and the terminators with lower free energy corelated with higher upstream gene expression. Meanwhile, the terminator with longer stem length, more compact loop and perfect U-tract structure was benefit to the transcription termination. Finally, a terminator strength classification model was established, and the verification experiment based on 20 synthetic terminators indicated that the model can distinguish strong and weak terminators to certain extent. The results help to elucidate the role of terminators in gene expression, and the key factors identified are crucial for rational design of terminators, and the model provided a method for terminator strength prediction.
原核生物中的内在终止子形成二级RNA结构并终止转录。然而，由于终止子强度不同，转录渗漏很常见。除了典型的发夹结构和U序列结构外，终止子的详细序列和热力学特征尚不完全清楚，其对上游基因表达的影响也不明确。因此，利用终止子进行更高精度的表达调控仍具有挑战性。在此，我们首先确定了包括间隔序列和终止子序列在内的3'端序列对上游和下游基因表达的影响。其次，构建了终止子突变文库，并使用FlowSeq技术分析了终止效率不同的热力学和序列特征。结果表明，在终止子调控下，上游和下游基因的表达呈负相关（r = -0.60），自由能较低的终止子与较高的上游基因表达相关。同时，茎长度更长、环更紧凑且具有完美U序列结构的终止子有利于转录终止。最后，建立了终止子强度分类模型，基于20个合成终止子的验证实验表明该模型在一定程度上能够区分强终止子和弱终止子。这些结果有助于阐明终止子在基因表达中的作用，所确定的关键因素对终止子的合理设计至关重要，且该模型为终止子强度预测提供了一种方法。
