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Regulation of Expression at Multiple Steps01:23

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The gene expression in cells is regulated at different stages: (i) transcription, (ii) RNA processing, (iii) RNA localization, and (iv) translation. Transcriptional regulation is mediated by regulatory proteins such as transcription factors, activators, or repressors—these control gene expression by initiating or inhibiting the transcription of genes. Once a precursor or pre-mRNA is produced, it undergoes post-transcriptional modification, including 5' capping, splicing, and the...
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Transcriptional Regulation: Riboswitches01:23

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Riboswitches are RNA elements that regulate gene expression by altering their secondary structures in response to specific effector molecules. These elements, located in the leader regions of certain mRNAs, act as transcriptional regulators by toggling between alternative conformations to control downstream gene expression. Riboswitch-mediated regulation is a precise mechanism for modulating biosynthetic pathways, as exemplified by the riboflavin biosynthesis pathway in Bacillus...
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Riboswitches01:56

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Riboswitches are non-coding mRNA domains that regulate the transcription and translation of downstream genes without the help of proteins. Riboswitches bind directly to a metabolite and can form unique stem-loop or hairpin structures in response to the amount of the metabolite present. They have two distinct regions – a metabolite-binding aptamer and an expression platform.
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Translational regulation in prokaryotes ensures efficient protein synthesis by controlling ribosome access to mRNA. This regulation is mediated by secondary RNA structures, including translational riboswitches, RNA thermometers, and small RNAs (sRNAs), which respond to intracellular and environmental signals to modulate gene expression.Translational RiboswitchesRiboswitches in the leader region of mRNAs can regulate translation by altering the accessibility of the Shine-Dalgarno (SD) sequence,...
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Gene expression can be regulated at almost every step from gene to protein. Transcription is the step that is most commonly regulated. This involves the binding of proteins to short regulatory sequences on the DNA. This association can either promote or inhibit the transcription of a gene associated with the respective sequence.
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De novo-designed translation-repressing riboregulators for multi-input cellular logic.

Jongmin Kim1,2, Yu Zhou3,4, Paul D Carlson5,6

  • 1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.

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Researchers developed novel synthetic RNA devices called toehold and three-way junction (3WJ) repressors. These riboregulators enable precise control over gene expression, offering new tools for complex synthetic biology applications.

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Area of Science:

  • Synthetic Biology
  • RNA Engineering
  • Molecular Biology

Background:

  • Synthetic biological circuits are crucial for advanced applications but face limitations like part variability and crosstalk.
  • Existing biological parts often exhibit unpredictable behavior, restricting the complexity and reliability of engineered systems.

Purpose of the Study:

  • To design and develop novel, high-performance synthetic RNA-based translational repressors.
  • To create modular riboregulators with sensing and logic capabilities for enhanced gene expression control.

Main Methods:

  • De novo RNA design was employed to create toehold and three-way junction (3WJ) repressors.
  • Automated forward engineering and SHAPE-Seq were utilized to optimize and validate repressor performance and mechanisms.
  • Integration of repressors into biological circuits for evaluating complex logic functions.

Main Results:

  • Developed two types of synthetic riboregulators: toehold and 3WJ repressors.
  • Achieved high repression levels (up to 300-fold) and orthogonality for up to 15 devices.
  • Demonstrated successful implementation of NAND and NOR logic gates and a complex four-input genetic circuit in Escherichia coli.

Conclusions:

  • Toehold and 3WJ repressors offer significant improvements in dynamic range, orthogonality, and sequence detection compared to existing parts.
  • These novel RNA tools provide enhanced capabilities for constructing sophisticated and reliable synthetic biological circuits.
  • The developed repressors represent valuable additions to the synthetic biology toolbox for diverse biotechnological applications.