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Engineering Transcriptional Interference through RNA Polymerase Processivity Control.

Nolan J O'Connor1, Antoni E Bordoy1, Anushree Chatterjee1,2,3

  • 1Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado 80303, United States.

ACS Synthetic Biology
|March 12, 2021
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Summary

Antisense transcription

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Genetics

Background:

  • Antisense transcription is common in nature and affects gene expression via transcriptional interference (TI).
  • RNA polymerase (RNAP) processivity is linked to antisense transcription but its role in TI is not well understood.

Purpose of the Study:

  • To investigate the influence of RNAP processivity on TI.
  • To explore methods for controlling TI for gene regulation and synthetic biology applications.

Main Methods:

  • Utilized three antitermination strategies to control RNAP processivity: bicyclomycin, phage protein Psu, and ribosome-RNAP coupling.
  • Quantified gene repression using these methods, observing up to 38-fold reduction.
  • Engineered genetic logic gates (NAND, NOR) by combining protein roadblocks with TI.

Main Results:

  • Demonstrated that controlling RNAP processivity is crucial for effective TI.
  • Achieved significant gene repression (38-fold) through engineered RNAP collisions and antisense RNA interference.
  • Successfully designed functional genetic NAND and NOR logic gates.

Conclusions:

  • RNAP processivity is a key factor in modulating transcriptional interference.
  • Transcriptional interference offers a powerful tool for gene regulation and synthetic biology design.
  • This work provides a foundation for developing novel genetic circuits and regulatory systems.