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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.
Highlights
From the ‡Department of Chemical and Biological Engineering and ¶Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208 and the §Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14850
We determined that an internal desthiobiotin-triethylene glycol modification positioned in the transcribed strand of in vitro transcription DNA templates efficiently and stably halts E. coli RNA polymerases (RNAPs)
E. coli RNAP has been reported previously to bypass abasic sites in the transcribed DNA strand [15], we envisioned that the desthiobiotin–TEG modification might enable protein-free transcription roadblocking at a defined DNA template position
Summary
From the ‡Department of Chemical and Biological Engineering and ¶Center for Synthetic Biology, Northwestern University, Evanston, Illinois 60208 and the §Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14850. We determined that an internal desthiobiotin-triethylene glycol (desthiobiotin–TEG) modification positioned in the transcribed strand of in vitro transcription DNA templates efficiently and stably halts E. coli RNAP.
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