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Zeitschriftenartikel

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

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons82132

Said,  Nelly
Max-Planck Research Group RNA Biology, Max Planck Institute for Infection Biology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons82120

Rieder,  Renate
Max-Planck Research Group RNA Biology, Max Planck Institute for Infection Biology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons81946

Hurwitz,  Robert
Core Facilities / Proteinpurification, Max Planck Institute for Infection Biology, Max Planck Society;

Urlaub,  Henning
Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons82198

Vogel,  Jörg
Max-Planck Research Group RNA Biology, Max Planck Institute for Infection Biology, Max Planck Society;

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Nucl_Acids_Res_2009_37_e133.pdf
(Verlagsversion), 7MB

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Zitation

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.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-000E-C070-A
Zusammenfassung
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.