Monitoring Translocation of Multisubunit RNA Polymerase Along the DNA with Fluorescent Base Analogues

  • Anssi M. Malinen
  • Matti Turtola
  • Georgiy A. Belogurov
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1276)

Abstract

Here we describe a direct fluorescence method that reports real-time occupancies of the pre- and post-translocated state of multisubunit RNA polymerase. In a stopped-flow setup, this method is capable of resolving a single base-pair translocation motion of RNA polymerase in real time. In a conventional spectrofluorometer, this method can be employed for studies of the time-averaged distribution of RNA polymerase on the DNA template. This method utilizes commercially available base analogue fluorophores integrated into template DNA strand in place of natural bases. We describe two template DNA strand designs where translocation of RNA polymerase from a pre-translocation to a post-translocation state results in disruption of stacking interactions of fluorophore with neighboring bases, with a concomitant large increase in fluorescence intensity.

Key words

Transcription RNA polymerase Translocation Fluorescence Base analogue Stopped-flow Rapid quench-flow 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Anssi M. Malinen
    • 1
  • Matti Turtola
    • 1
  • Georgiy A. Belogurov
    • 1
  1. 1.Department of BiochemistryUniversity of TurkuTurkuFinland

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