Analysis of duplex unwinding by RNA helicases using stopped-flow fluorescence spectroscopy

Andrea Putnam, Eckhard Jankowsky

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The characterization of unwinding reactions by RNA helicases often requires the determination of rate constants that are too fast to be measured by traditional, manual gel-based methods. Stopped-flow fluorescence measurements allow access to fast unwinding rate constants. In this chapter, we outline strategies and experimental considerations for the design of stopped-flow fluorescence experiments to monitor duplex unwinding by RNA helicases, with focus on DEAD-box helicases. We discuss advantages, disadvantages, and technical considerations for stopped-flow approaches, as well as substrate design. In addition, we list protocols and explain functional information obtained with these experiments.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages1-27
Number of pages27
DOIs
StatePublished - 2012
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume511
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • ATP
  • Base pair
  • Fluorescence
  • Helix
  • Kinetics
  • RNA
  • Stopped flow

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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