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.
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