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Methods Enzymol. 2012;511:111-30. doi: 10.1016/B978-0-12-396546-2.00005-X.

RNA catalysis as a probe for chaperone activity of DEAD-box helicases.

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  • 1Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.

Abstract

DEAD-box proteins are vitally important to cellular processes and make up the largest class of helicases. Many DEAD-box proteins function as RNA chaperones by accelerating structural transitions of RNA, which can result in the resolution of misfolded conformers or conversion between functional structures. While the biological importance of chaperone proteins is clear, their mechanisms are incompletely understood. Here, we illustrate how the catalytic activity of certain RNAs can be used to measure RNA chaperone activity. By measuring the amount of substrate converted to product, the fraction of catalytically active molecules is measured over time, providing a quantitative measure of the formation or loss of native RNA. The assays are described with references to group I and group II introns and their ribozyme derivatives, and examples are included that illustrate potential complications and indicate how catalytic activity measurements can be combined with physical approaches to gain insights into the mechanisms of DEAD-box proteins as RNA chaperones.

Copyright © 2012 Elsevier Inc. All rights reserved.

PMID:
22713317
[PubMed - indexed for MEDLINE]
PMCID:
PMC3399669
Free PMC Article
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