title: Assessing RNA interactions with proteins by DRaCALA.
abstract: Discovery of RNA elements, including riboswitches and regulatory RNAs, has revealed additional regulatory mechanisms for transcript stability, transcript termination, and translational initiation. These regulatory RNA molecules act through direct binding to cellular targets including other RNA molecules, proteins, and low molecular weight metabolites. RNA-RNA interactions based on complementarity can be identified through bioinformatic analysis. However, identification of novel interactions between these regulatory RNA molecules and their partners other than complementary sequences is more challenging. We have developed a technique called Differential Radial Capillary Action of Ligand Assay (DRaCALA) to facilitate the detection of direct binding between RNA elements to proteins or low molecular weight ligands. Previously, we have described the adaptation of this technique to detect the binding interaction between Vc2 riboswitch to a signaling cyclic dinucleotide called cyclic-di-GMP. Here, we describe the adaptation of DRaCALA for identifying sequence-specific RNA-binding proteins directly from E. coli cell lysates expressing the recombinant binding protein. DRaCALA can be used to qualitatively and quantitatively assess RNA-protein interaction in whole cell lysate, determine the kinetics of the binding, and test for competitors. Using DRaCALA in a high-throughput format has the potential to rapidly identify sequence-specific RNA-binding proteins.
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA.
authors.forename: Darshan K
authors.initials: DK
authors.lastname: Patel
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, USA; Maryland Pathogen Research Institute, University of Maryland, College Park, MD, USA.
authors.forename: Margo P
authors.initials: MP
authors.lastname: Gebbie
authors.affiliation: Maryland Pathogen Research Institute, University of Maryland, College Park, MD, USA; Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, USA. Electronic address: vtlee@umd.edu.
authors.forename: Vincent T
authors.initials: VT
authors.lastname: Lee
mesh_terms: D001671:Biochemistry / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D011485:Protein Binding; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D058928:Riboswitch
chemical_list: C093587:CsrA protein, E coli; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D058928:Riboswitch
keywords: DRaCALA; High throughput screen; Protein-ligand interaction; RNA-protein interaction; Regulatory RNA; Riboswitch; Small RNA