Real-time observation of backtracking by bacterial RNA polymerase
| Autor: | Tomasz Heyduk, Agnieszka Lass-Napiorkowska |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
DNA Bacterial biology Transcription Genetic Backtracking Escherichia coli Proteins RNA DNA-Directed RNA Polymerases Biochemistry Molecular biology Article 03 medical and health sciences chemistry.chemical_compound RNA Bacterial 030104 developmental biology chemistry Transcription (biology) RNA polymerase Coding strand biology.protein Biophysics Escherichia coli AP site Polymerase DNA |
| Popis: | RNA polymerase (RNAP) backtracking is a backward sliding of the enzyme along DNA and RNA. It plays important roles in many essential processes in bacteria and in eukaryotes. We describe here a fluorescence-based approach that allows a real-time observation of bacterial RNAP backtracking. A Cy3 fluorescence probe, when incorporated into a specific site in the nontemplate strand near the site of backtracking, allows RNAP movements to be monitored near the probe because of a robust enhancement of fluorescence caused by protein proximity. Using this approach, we showed that binding of NTP to the active site prior to phosphodiester bond formation inhibited backtracking, consistent with the coupling of NTP binding to translocation. The extent and the kinetics of backtracking did not show a simple correlation with the instability of the DNA-RNA hybrid, indicating a more complex dependence of backtracking on DNA template sequence. Experiments with transcription through an abasic site in DNA template or neutravidin bound to biotinylated template strand base illustrated an important role of backtracking in defining how RNAP reacts to such obstacles in the DNA template. The described approach will be a useful tool in deciphering the mechanism of backtracking and in studying factors that affect the backtracking. |
| Databáze: | OpenAIRE |
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