Effect of terminators on the downstream transcript unit with gene expression in <italic style="font-style: italic">Escherichia</italic> <italic style="font-style: italic">coli</italic>

REN Jiawei; ZHANG Jinpeng; XU Guoqiang; ZHANG Xiaomei; XU Zhenghong; ZHANG Xiaojuan

doi:10.12211/2096-8280.2024-046

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Effect of terminators on the downstream transcript unit with gene expression in Escherichia coli

Research Article | 更新时间：2025-06-20

- Effect of terminators on the downstream transcript unit with gene expression in Escherichia coli
- Synthetic Biology Journal Vol. 6, Issue 1, Pages: 213-227(2025)
- 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：
- 基金信息：
- DOI：10.12211/2096-8280.2024-046
  CLC： Q819
- Received：17 June 2024，
  
  Revised：2024-08-22，
  
  Published：31 January 2025
- 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

LAURSEN B S , SØRENSEN H P , MORTENSEN K K , et al . Initiation of protein synthesis in bacteria [J ] . Microbiology and Molecular Biology Reviews , 2005 , 69 ( 1 ): 101 - 123 .

MORENO J M , SØRENSEN H P , MORTENSEN K K , et al . Macromolecular mimicry in translation initiation: a model for the initiation factor IF2 on the ribosome [J ] . IUBMB Life , 2000 , 50 ( 6 ): 347 - 354 .

RAMAKRISHNAN V . Ribosome structure and the mechanism of translation [J ] . Cell , 2002 , 108 ( 4 ): 557 - 572 .

ZHOU S H , DU G C , KANG Z , et al . The application of powerful promoters to enhance gene expression in industrial microorganisms [J ] . World Journal of Microbiology & Biotechnology , 2017 , 33 ( 2 ): 23 .

XU N , WEI L , LIU J . Recent advances in the applications of promoter engineering for the optimization of metabolite biosynthesis [J ] . World Journal of Microbiology & Biotechnology , 2019 , 35 ( 2 ): 33 .

ZALATAN F , PLATT T . Effects of decreased cytosine content on rho interaction with the rho-dependent Terminator trp t ′ in Escherichia coli [J ] . Journal of Biological Chemistry , 1992 , 267 ( 27 ): 19082 - 19088 .

HENKIN T M , YANOFSKY C . Regulation by transcription attenuation in bacteria: how RNA provides instructions for transcription termination/antitermination decisions [J ] . BioEssays , 2002 , 24 ( 8 ): 700 - 707 .

ABE H , ABO T , AIBA H . Regulation of intrinsic Terminator by translation in Escherichia coli : transcription termination at a distance downstream [J ] . Genes to Cells , 1999 , 4 ( 2 ): 87 - 97 .

RAY-SONI A , BELLECOURT M J , LANDICK R . Mechanisms of bacterial transcription termination: all good things must end [J ] . Annual Review of Biochemistry , 2016 , 85 : 319 - 347 .

LEE D N , PHUNG L , STEWART J , et al . Transcription pausing by Escherichia coli RNA polymerase is modulated by downstream DNA sequences [J ] . Journal of Biological Chemistry , 1990 , 265 ( 25 ): 15145 - 15153 .

KIREEVA M L , KASHLEV M . Mechanism of sequence-specific pausing of bacterial RNA polymerase [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2009 , 106 ( 22 ): 8900 - 8905 .

CHAN C L , WANG D G , LANDICK R . Multiple interactions stabilize a single paused transcription intermediate in which hairpin to 3΄ end spacing distinguishes pause and termination pathways [J ] . Journal of Molecular Biology , 1997 , 268 ( 1 ): 54 - 68 .

MAIRHOFER J , WITTWER A , CSERJAN-PUSCHMANN M , et al . Preventing T7 RNA polymerase read-through transcription-a synthetic termination signal capable of improving bioprocess stability [J ] . ACS Synthetic Biology , 2015 , 4 ( 3 ): 265 - 273 .

ZHANG B , YU M , ZHOU Y , et al . Improvement of L-ornithine production by attenuation of argF in engineered Corynebacterium glutamicum S9114 [J ] . AMB Express , 2018 , 8 ( 1 ): 26 .

JU X W , LI D Y , LIU S X . Full-length RNA profiling reveals pervasive bidirectional transcription terminators in bacteria [J ] . Nature Microbiology , 2019 , 4 ( 11 ): 1907 - 1918 .

AHN J H , KANG T J , KIM D M . Tuning the expression level of recombinant proteins by modulating mRNA stability in a cell-free protein synthesis system [J ] . Biotechnology and Bioengineering , 2008 , 101 ( 2 ): 422 - 427 .

CHEN Y J , LIU P , NIELSEN A A K , et al . Characterization of 582 natural and synthetic terminators and quantification of their design constraints [J ] . Nature Methods , 2013 , 10 ( 7 ): 659 - 664 .

CUI W J , LIN Q , HU R C , et al . Data-driven and in silico -assisted design of broad host-range minimal intrinsic terminators adapted for bacteria [J ] . ACS Synthetic Biology , 2021 , 10 ( 6 ): 1438 - 1450 .

ZHAI W J , DUAN Y T , ZHANG X M , et al . Sequence and thermodynamic characteristics of terminators revealed by FlowSeq and the discrimination of terminators strength [J ] . Synthetic and Systems Biotechnology , 2022 , 7 ( 4 ): 1046 - 1055 .

翟伟绩 . 大肠杆菌终止子文库构建及构效关系分析 [D ] . 无锡 : 江南大学 , 2022 .

ZHAI W J . The construction of the terminator library and analysis of the structure-activity relationship [D ] . Wuxi : Jiangnan University , 2022 .

DU L P , GAO R , FORSTER A C . Engineering multigene expression in vitro and in vivo with small terminators for T7 RNA polymerase [J ] . Biotechnology and Bioengineering , 2009 , 104 ( 6 ): 1189 - 1196 .

TARNOWSKI M J , GOROCHOWSKI T E . Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing [J ] . Nature Communications , 2022 , 13 ( 1 ): 434 .

LI R , ZHANG Q , LI J B , et al . Effects of cooperation between translating ribosome and RNA polymerase on termination efficiency of the rho-independent terminator [J ] . Nucleic Acids Research , 2016 , 44 ( 6 ): 2554 - 2563 .

LALANNE J B , TAGGART J C , GUO M S , et al . Evolutionary convergence of pathway-specific enzyme expression stoichiometry [J ] . Cell , 2018 , 173 ( 3 ): 749 - 761.e38 .

GOROCHOWSKI T E , ESPAH BORUJENI A , PARK Y , et al . Genetic circuit characterization and debugging using RNA-seq [J ] . Molecular Systems Biology , 2017 , 13 ( 11 ): 952 .

KOSURI S , GOODMAN D B , CAMBRAY G , et al . Composability of regulatory sequences controlling transcription and translation in Escherichia coli [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2013 , 110 ( 34 ): 14024 - 14029 .

CAMBRAY G , GUIMARAES J C , ARKIN A P . Evaluation of 244,000 synthetic sequences reveals design principles to optimize translation in Escherichia coli [J ] . Nature Biotechnology , 2018 , 36 : 1005 - 1015 .

EVFRATOV S A , OSTERMAN I A , KOMAROVA E S , et al . Application of sorting and next generation sequencing to study 5′-UTR influence on translation efficiency in Escherichia coli [J ] . Nucleic Acids Research , 2017 , 45 ( 6 ): 3487 - 3502 .

YOON B J . Hidden Markov models and their applications in biological sequence analysis [J ] . Current Genomics , 2009 , 10 ( 6 ): 402 - 415 .

KUO S T , JAHN R L , CHENG Y J , et al . Global fitness landscapes of the Shine-Dalgarno sequence [J ] . Genome Research , 2020 , 30 ( 5 ): 711 - 723 .

OSTERMAN I A , CHERVONTSEVA Z S , EVFRATOV S A , et al . Translation at first sight: the influence of leading codons [J ] . Nucleic Acids Research , 2020 , 48 ( 12 ): 6931 - 6942 .

VERMA M , CHOI J , COTTRELL K A , et al . A short translational ramp determines the efficiency of protein synthesis [J ] . Nature Communications , 2019 , 10 ( 1 ): 5774 .

GAO R , YU K , NIE J K , et al . Deep sequencing reveals global patterns of mRNA recruitment during translation initiation [J ] . Scientific Reports , 2016 , 6 : 30170 .

LESNIK E A , SAMPATH R , LEVENE H B , et al . Prediction of rho-independent transcriptional terminators in Escherichia coli [J ] . Nucleic Acids Research , 2001 , 29 ( 17 ): 3583 - 3594 .

DUAN Y T , ZHAI W J , LIU W J , et al . Fine-tuning multi-gene clusters via well-characterized gene expression regulatory elements: case study of the arginine synthesis pathway in C. glutamicum [J ] . ACS Synthetic Biology , 2021 , 10 ( 1 ): 38 - 48 .

HUDSON A J , WIEDEN H J . Rapid generation of sequence-diverse terminator libraries and their parameterization using quantitative Term-Seq [J ] . Synthetic Biology , 2019 , 4 ( 1 ): ysz026 .

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Related Institution

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⁰

Effect of terminators on the downstream transcript unit with gene expression in Escherichia coli

DOI：10.12211/2096-8280.2024-046

摘要

Abstract

关键词

Keywords

references