Abstract
Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current in vitro transcription “roadblocking” approaches inhibit transcription elongation by blocking RNAP with a protein bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires inclusion of a protein factor extrinsic to the minimal in vitro transcription reaction. In this work, we developed a chemical approach for halting transcription by Escherichia coli RNAP. We first established a sequence-independent method for site-specific incorporation of chemical lesions into dsDNA templates by sequential PCR and translesion synthesis. We then show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification or 1,N6-etheno-2'-deoxyadenosine base efficiently and stably halts Escherichia coli RNAP transcription. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables display of nascent RNA molecules from RNAP in a minimal in vitro transcription reaction.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 6401-6412 |
| Number of pages | 12 |
| Journal | Journal of Biological Chemistry |
| Volume | 295 |
| Issue number | 19 |
| DOIs | |
| State | Published - May 8 2020 |
Funding
This work was supported by an Arnold O. Beckman postdoctoral fellowship (to E. J. S.), NIGMS, National Institutes of Health Grant GM025232 (to J. T. L.)., and Searle Funds at The Chicago Community Trust (to J. B. L.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
ASJC Scopus subject areas
- Molecular Biology
- Biochemistry
- Cell Biology