大肠杆菌中终止子对下游转录单元基因表达的影响

任家卫; 张金鹏; 徐国强; 张晓梅; 许正宏; 张晓娟

doi:10.12211/2096-8280.2024-046

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大肠杆菌中终止子对下游转录单元基因表达的影响

研究论文 | 更新时间：2025-06-20

- 大肠杆菌中终止子对下游转录单元基因表达的影响
- Effect of terminators on the downstream transcript unit with gene expression in Escherichia coli
- 合成生物学 2025年6卷第1期页码：213-227
- 作者机构：
  
  1.江南大学生物工程学院工业生物技术教育部重点实验室，江苏无锡 214122
  2.江南大学粮食发酵与食品生物制造国家工程研究中心，江苏无锡 214122
  3.江南大学生命科学与健康工程学院，江苏无锡 214122
  4.四川大学轻工科学与工程学院，四川成都 610065
- 作者简介：
  
  [ "任家卫（1999—），男，硕士研究生。研究方向为终止子调控元件开发。E-mail：297887816@qq.com" ]
  [ "张晓娟（1982—），女，教授。研究方向为酿造食品微生物开发与应用、新型工业菌株的基因表达调控元件开发。E-mail：zhangxj@jiangnan.edu.cn" ]
- 基金信息：
  
  国家自然科学基金(32171421)
- DOI：10.12211/2096-8280.2024-046
  中图分类号： Q819
- 收稿：2024-06-17，
  
  修回：2024-08-22，
  
  纸质出版：2025-01-31
- 稿件说明：
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任家卫，张金鹏，徐国强，张晓梅，许正宏，张晓娟. 大肠杆菌中终止子对下游转录单元基因表达的影响［J］. 合成生物学， 2025， 6（1）： 213-227

REN Jiawei， ZHANG Jinpeng， XU Guoqiang， ZHANG Xiaomei， XU Zhenghong， ZHANG Xiaojuan. Effect of terminators on the downstream transcript unit with gene expression in Escherichia coli［J］. Synthetic Biology Journal， 2025， 6（1）： 213-227
任家卫，张金鹏，徐国强，张晓梅，许正宏，张晓娟. 大肠杆菌中终止子对下游转录单元基因表达的影响［J］. 合成生物学， 2025， 6（1）： 213-227 DOI： 10.12211/2096-8280.2024-046.

REN Jiawei， ZHANG Jinpeng， XU Guoqiang， ZHANG Xiaomei， XU Zhenghong， ZHANG Xiaojuan. Effect of terminators on the downstream transcript unit with gene expression in Escherichia coli［J］. Synthetic Biology Journal， 2025， 6（1）： 213-227 DOI： 10.12211/2096-8280.2024-046.

摘要

在基因转录过程中，RNA聚合酶通过识别启动子序列启动转录过程，而当其识别到位于3′-UTR的终止子序列后，转录复合物解离，转录过程终止。因此，转录单元内的启动子和终止子分别发挥启动和终止转录的作用。然而，对于下游的转录单元，终止子除了终止转录本通读这一直接作用以外，其与RNA聚合酶之间的解离可能会影响后续转录单元中启动子与RNA聚合酶的结合，从而间接改变下游转录单元的表达。这种跨转录单元的终止子和启动子之间的互作关系目前尚缺少研究，因此，明确终止子对下游转录单元的转录反应强度的影响对于精准调控基因表达、开发高效终止子具有重要的意义。本研究通过one-pot技术将9种终止子、5种间隔序列和9种启动子进行组合构建了一个包含405种不同组合元件（终止子-间隔序列-启动子）的组装文库，基于FlowSeq技术对文库中所有组合进行测序和荧光强度分析，进而建立组合序列-下游基因表达之间的相关性。结果表明弱终止子、短间隔以及强启动子的组合更有利于提高下游基因的表达。通过对转录本的定量分析发现弱终止子不仅提高下游的渗漏转录（提高21～70倍），同时也促进了下游启动子重新招募RNA聚合酶进行重启转录（提高2～3倍）。本研究解析了终止子对下游转录单元基因表达的调控效果和机制，为利用终止子构建基因回路提供设计依据。

Abstract

During gene transcription

RNA polymerase initiates the process by recognizing the promoter sequence

and terminates it upon recognizing the terminator sequence located at the 3′-UTR

leading to dissociation of the transcription complex. Therefore

promoters and terminators within the transcription unit play the role of initiating and terminating transcription

respectively. For downstream transcription units

in addition to the direct effect of terminating transcript read-through

the dissociation of the RNA polymerase from the terminator may affect the binding of the promoter to RNA polymerase in the subsequent transcription unit

thus indirectly altering the expression of the downstream transcription unit. This interplay between terminators and promoters across transcription units remains poorly understood

therefore

elucidating the impact of terminators on the transcriptional strength of downstream transcription units is of great significance for the precise regulation of gene expression and the development of efficient terminators. In this study

a library containing 405 different combinatorial elements (terminator-spacer-promoter) was constructed by combining nine terminators

five spacer sequences

and nine promoters using one-pot assembly technology. All combinations in the library were sequenced and analyzed in terms of fluorescence intensity based on the FlowSeq technology to establish the correlations between combinatorial sequences and downstream gene expression. The results showed that combinations of weak terminators

short spacers

and strong terminators were more favorable to enhance the expression of downstream genes

while combinations of strong terminators

long spacers

and weak terminators reduced the expression of downstream genes. Quantitative analysis of transcription revealed that weak terminators not only enhanced downstream leakage transcription (21~70-fold enhancement)

but also facilitated downstream promoters to re-recruit RNA polymerase for re-promoted transcription (2~3-fold enhancement). This study has elucidated the effect and mechanism of terminators on the regulation of gene expression in the downstream transcription units

providing a design framework for the construction of gene circuits using terminators.

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references

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