Sequence and thermodynamic characteristics of terminators revealed by FlowSeq and the discrimination of terminators strength

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 E. Coli, 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.