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

Commun Biol. 2022 May 12;5(1):457. doi: 10.1038/s42003-022-03382-0.

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.

MeSH terms

  • DNA-Directed RNA Polymerases / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins* / genetics
  • Escherichia coli Proteins* / metabolism
  • Nucleic Acid Conformation
  • Transcription, Genetic*

Substances

  • Escherichia coli Proteins
  • DNA-Directed RNA Polymerases