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Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA.

Authors
Type
Published Article
Journal
RNA
1469-9001
Publisher
Cold Spring Harbor Laboratory
Publication Date
Volume
13
Issue
12
Pages
2175–2188
Identifiers
PMID: 17959928
Source
Medline
License
Unknown

Abstract

RNA unfolding and folding reactions in physiological conditions can be facilitated by mechanical force one molecule at a time. By using force-measuring optical tweezers, we studied the mechanical unfolding and folding of a hairpin-type pseudoknot in human telomerase RNA in a near-physiological solution, and at room temperature. Discrete two-state folding transitions of the pseudoknot are seen at approximately 10 and approximately 5 piconewtons (pN), with ensemble rate constants of approximately 0.1 sec(-1), by stepwise force-drop experiments. Folding studies of the isolated 5'-hairpin construct suggested that the 5'-hairpin within the pseudoknot forms first, followed by formation of the 3'-stem. Stepwise formation of the pseudoknot structure at low forces are in contrast with the one-step unfolding at high forces of approximately 46 pN, at an average rate of approximately 0.05 sec(-1). In the constant-force folding trajectories at approximately 10 pN and approximately 5 pN, transient formation of nonnative structures were observed, which is direct experimental evidence that folding of both the hairpin and pseudoknot takes complex pathways. Possible nonnative structures and folding pathways are discussed.

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