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

被引:67
作者
Said, Nelly [1 ]
Rieder, Renate [1 ]
Hurwitz, Robert [2 ]
Deckert, Jochen [3 ,4 ]
Urlaub, Henning [5 ]
Vogel, Joerg [1 ]
机构
[1] Max Planck Inst Infect Biol, RNA Biol Grp, D-10117 Berlin, Germany
[2] Max Planck Inst Infect Biol, Prot Purificat Facil, D-10117 Berlin, Germany
[3] Dept Cellular Biochem, D-95326 Kulmbach, Germany
[4] Roche Kulmbach GmbH, D-95326 Kulmbach, Germany
[5] Max Planck Inst Biophys Chem, Bioanalyt Mass Spectrometry Grp, D-37077 Gottingen, Germany
关键词
SMALL NONCODING RNAS; MESSENGER-RNA; ESCHERICHIA-COLI; AFFINITY PURIFICATION; THERMODYNAMIC ANALYSIS; PROTEIN-COMPOSITION; COAT PROTEIN; 6S RNA; BINDING; IDENTIFICATION;
D O I
10.1093/nar/gkp719
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:e133 / e133
页数:14
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