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Nucleic Acids Research Advance Access originally published online on August 11, 2009
Nucleic Acids Research 2009 37(18):6205-6213; doi:10.1093/nar/gkp646
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Nucleic Acids Research, 2009, Vol. 37, No. 18 6205-6213
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

Effect of salt and RNA structure on annealing and strand displacement by Hfq

Julia F. Hopkins1, Subrata Panja2, Stephanie A. N. McNeil3 and Sarah A. Woodson2,*

1Program in Cellular, Molecular and Developmental Biology and Biophysics, 2T.C. Jenkins Department of Biophysics and 3JHU-NIH Graduate Partnership Program, Johns Hopkins University, 3400 N. Charles St, Baltimore, MD 21218, USA

*To whom correspondence should be addressed. Tel: +1 410 516 2015; Fax: +1 410 516 4118; Email: swoodson{at}jhu.edu

Received March 19, 2009. Revised June 3, 2009. Accepted July 20, 2009.

The Sm-like protein Hfq promotes the association of small antisense RNAs (sRNAs) with their mRNA targets, but the mechanism of Hfq's RNA chaperone activity is unknown. To investigate RNA annealing and strand displacement by Hfq, we used oligonucleotides that mimic functional sequences within DsrA sRNA and the complementary rpoS mRNA. Hfq accelerated at least 100-fold the annealing of a fluorescently labeled molecular beacon to a 16-nt RNA. The rate of strand exchange between the oligonucleotides increased 80-fold. Therefore, Hfq is very active in both helix formation and exchange. However, high concentrations of Hfq destabilize the duplex by preferentially binding the single-stranded RNA. RNA binding and annealing were completely inhibited by 0.5 M salt. The target site in DsrA sRNA was 1000-fold less accessible to the molecular beacon than an unstructured oligonucleotide, and Hfq accelerated annealing with DsrA only 2-fold. These and other results are consistent with recycling of Hfq during the annealing reaction, and suggest that the net reaction depends on the relative interaction of Hfq with the products and substrates.


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