Chapter title |
Use of Aptamer Tagging to Identify In Vivo Protein Binding Partners of Small Regulatory RNAs.
|
---|---|
Chapter number | 11 |
Book title |
Bacterial Regulatory RNA
|
Published in |
Methods in molecular biology, January 2012
|
DOI | 10.1007/978-1-61779-949-5_11 |
Pubmed ID | |
Book ISBNs |
978-1-61779-948-8, 978-1-61779-949-5
|
Authors |
Colin P. Corcoran, Renate Rieder, Dimitri Podkaminski, Benjamin Hofmann, Jörg Vogel |
Abstract |
Small regulatory RNAs (sRNAs) are short, generally noncoding RNAs that act posttranscriptionally to control target gene expression. Over the past 10 years there has been a rapid expansion in the discovery and characterization of sRNAs in a diverse range of bacteria. Paradigm shifts in our understanding of the breadth of posttranscriptional control by sRNAs were achieved in a number of pioneering studies that involved immunoprecipitation of a known RNA chaperone, the near-ubiquitous Hfq, followed by sequencing to identify novel putative regulators and targets. To perform the converse experiment, we previously developed a method which uses an aptamer-tagged sRNA to allow purification of in vivo assembled RNA-protein complexes and subsequent identification of bound proteins. We successfully implemented this protocol using the Hfq-associated sRNA, InvR, tagged with a tandem repeat of the commonly used MS2-aptamer. Incorporation of the aptamer had no effect on sRNA stability or activity. InvR-MS2 could be effectively purified along with associated proteins, such as Hfq, using maltose binding protein fused to the MS2 coat protein (MBP-MS2) immobilized on an amylose column. Mass-spectroscopy was also used to identify previously uncharacterized protein partners. These results have been described previously (Said et al., Nucleic Acids Res 37:e133, 2009) and thus the figures presented here are intended solely as an illustrative guide to complement this detailed step-by-step protocol. |
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