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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
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T7 RNA polymerases backed up by covalently trapped proteins catalyze highly error prone transcription

Toshiaki Nakano1, Ryo Ouchi, Junya Kawazoe

  • 1Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.

The Journal of Biological Chemistry
|January 12, 2012
PubMed

Insights

DNA-protein cross-links (DPCs) block transcription by RNA polymerases (RNAPs). Stalled RNAPs encountering DPCs become error-prone, causing mutations, but trailing RNAPs can promote bypass of these roadblocks.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • RNA polymerases (RNAPs) navigate DNA-bound proteins during transcription.
  • DNA-protein cross-links (DPCs) can form when proteins are covalently trapped on DNA.
  • The impact of DPCs on transcription is not well understood.

Purpose of the Study:

  • To investigate how DNA-protein cross-links (DPCs) affect in vitro transcription.
  • To analyze the fidelity of RNA polymerases (RNAPs) encountering DPCs and other stalled RNAPs.
  • To explore the interactions between RNAPs when stalled by persistent DNA roadblocks.

Main Methods:

  • Construction of DNA templates containing site-specific DPCs.
  • In vitro transcription assays using phage T7 RNA polymerase (RNAP).
  • Sequence analysis of RNA transcripts to detect mutations.

Main Results:

  • DPCs act as significant, though not absolute, blocks to T7 RNAP transcription.
  • Stalled RNAPs, both at DPCs and other stalled RNAPs, exhibit high error rates, inducing mutations in upstream DNA.
  • Trailing RNAPs can stimulate forward translocation of stalled RNAPs, facilitating DPC bypass.

Conclusions:

  • DPCs significantly impact transcriptional fidelity, leading to mutations in previously intact DNA regions.
  • RNAP-RNAP interactions play a crucial role in overcoming persistent DNA roadblocks like DPCs.
  • This study provides novel insights into transcriptional accuracy and RNAP behavior at stalled sites.

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