Chemical roadblocking of DNA transcription for nascent RNA display.
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.
['Strobel|Eric J|EJ|0000-0002-7082-2881', 'Lis|John T|JT|0000-0003-1201-9406', 'Lucks|Julius B|JB|0000-0002-0619-6505']
D004247:DNA D012321:DNA-Directed RNA Polymerases D004926:Escherichia coli D029968:Escherichia coli Proteins D012313:RNA D013698:Templates, Genetic D014158:Transcription, Genetic
transcription RNA RNA polymerase translesion synthesis RNA folding RNAP roadblocking biotin desthiobiotin etheno-dA site-specific transcription arrest