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Nucleic Acids Research Advance Access published online on September 2, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp719
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© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

In vivo expression and purification of aptamer-tagged small RNA regulators

Nelly Said1, Renate Rieder1, Robert Hurwitz2, Jochen Deckert3,4, Henning Urlaub5 and Jörg Vogel1,*

1RNA Biology Group, 2Protein Purification Facility, Max Planck Institute for Infection Biology, Charitéplatz 1, 10117 Berlin, 3Department of Cellular Biochemistry, 4Roche Kulmbach GmbH, 95326 Kulmbach, and 5Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany

*To whom correspondence should be addressed. Tel: +49-30-28460-265; Fax: +49-30-28460-244; Email: vogel{at}mpiib-berlin.mpg.de

Received July 6, 2009. Revised July 31, 2009. Accepted August 14, 2009.

Small non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators of bacterial gene expression. To study sRNAs and their potential protein interaction partners, it is desirable to purify sRNAs from cells in their native form. Here, we used RNA-based affinity chromatography to purify sRNAs following their expression as aptamer-tagged variants in vivo. To this end, we developed a family of plasmids to express sRNAs with any of three widely used aptamer sequences (MS2, boxB, eIF4A), and systematically tested how the aptamer tagging impacted on intracellular accumulation and target regulation of the Salmonella GcvB, InvR or RybB sRNAs. In addition, we successfully tagged the chromosomal rybB gene with MS2 to observe that RybB-MS2 is fully functional as an envelope stress-induced repressor of ompN mRNA following induction of sigmaE. We further demonstrate that the common sRNA-binding protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. The presented affinity purification strategy may facilitate the isolation of in vivo assembled sRNA–protein complexes in a wide range of bacteria.


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