Site-specific photolabile roadblocks for the study of transcription elongation in biologically complex systems

Abstract

Transcriptional pausing is crucial for the timely expression of genetic information. Biochemical methods quantify the half-life of paused RNA polymerase (RNAP) by monitoring restarting complexes across time. However, this approach may produce apparent half-lives that are longer than true pause escape rates in biological contexts where multiple consecutive pause sites are present. We show here that the 6-nitropiperonyloxymethyl (NPOM) photolabile group provides an approach to monitor transcriptional pausing in biological systems containing multiple pause sites. We validate our approach using the well-studied his pause and show that an upstream RNA sequence modulates the pause half-life. NPOM was also used to study a transcriptional region within the Escherichia coli thiC riboswitch containing multiple consecutive pause sites. We find that an RNA hairpin structure located upstream to the region affects the half-life of the 5′ most proximal pause site—but not of the 3′ pause site—in contrast to results obtained using conventional approaches not preventing asynchronous transcription. Our results show that NPOM is a powerful tool to study transcription elongation dynamics within biologically complex systems.