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

Said, N., Rieder, R., Hurwitz, R., Deckert, J., Urlaub, H., & Vogel, J. (2009). In vivo expression and purification of aptamer-tagged small RNA regulators. Nucleic Acids Research, 37(20): e133.

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Nucl_Acids_Res_2009_37_e133-1.pdf (Publisher version), 7MB
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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.
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 Creators:
Said, Nelly1, Author           
Rieder, Renate1, Author           
Hurwitz, Robert2, Author           
Deckert, Jochen, Author
Urlaub, Henning3, Author
Vogel, Jörg1, Author           
Affiliations:
1Max-Planck Research Group RNA Biology, Max Planck Institute for Infection Biology, Max Planck Society, ou_1664150              
2Core Facilities / Proteinpurification, Max Planck Institute for Infection Biology, Max Planck Society, ou_1664144              
3Max Planck Society, ou_persistent13              

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 Abstract: 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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Language(s): eng - English
 Dates: 2009-11
 Publication Status: Issued
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 Rev. Type: Peer
 Identifiers: eDoc: 572817
ISI: 000271819900032
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Title: Nucleic Acids Research
Source Genre: Journal
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Pages: - Volume / Issue: 37 (20) Sequence Number: e133 Start / End Page: - Identifier: ISSN: 0305-1048