Biomaterial Database

Probing Hfq:RNA interactions with hydroxyl radical and RNase footprinting. 2015

Michael J Ellis, and Ryan S Trussler, and Joseph A Ross, and David B Haniford

Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, 1151 Richmond St., London, ON, Canada, N6A 5C1.

RNA footprinting and structure probing techniques are used to characterize the interaction between RNA-binding proteins and RNAs in vitro. Hydroxyl radical footprinting results in the identification of protein binding site(s) in an RNA. Ribonuclease (RNase) structure probing is a complementary technique that also provides information about protein binding sites, as well as RNA structure and possible protein-directed RNA remodeling. Here we provide a comprehensive protocol for studying the interaction between Hfq and an mRNA or sRNA of interest using a combination of RNase A, T1, and V1 as well as hydroxyl radical footprinting techniques. Detailed protocols for in vitro synthesis of (32)P-labeled RNA; formation of Hfq:RNA binary complex(es), RNase, and hydroxyl radical footprinting; preparation and running of sequencing gels; and data analysis are provided.

UI	MeSH Term	Description	Entries
D009690	Nucleic Acid Conformation	The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.	DNA Conformation,RNA Conformation,Conformation, DNA,Conformation, Nucleic Acid,Conformation, RNA,Conformations, DNA,Conformations, Nucleic Acid,Conformations, RNA,DNA Conformations,Nucleic Acid Conformations,RNA Conformations
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D012329	RNA, Bacterial	Ribonucleic acid in bacteria having regulatory and catalytic roles as well as involvement in protein synthesis.	Bacterial RNA
D016601	RNA-Binding Proteins	Proteins that bind to RNA molecules. Included here are RIBONUCLEOPROTEINS and other proteins whose function is to bind specifically to RNA.	Double-Stranded RNA-Binding Protein,Double-Stranded RNA-Binding Proteins,ds RNA-Binding Protein,RNA-Binding Protein,ds RNA-Binding Proteins,Double Stranded RNA Binding Protein,Double Stranded RNA Binding Proteins,Protein, Double-Stranded RNA-Binding,Protein, ds RNA-Binding,RNA Binding Protein,RNA Binding Proteins,RNA-Binding Protein, Double-Stranded,RNA-Binding Protein, ds,RNA-Binding Proteins, Double-Stranded,ds RNA Binding Protein
D017665	Hydroxyl Radical	The univalent radical OH. Hydroxyl radical is a potent oxidizing agent.
D029968	Escherichia coli Proteins	Proteins obtained from ESCHERICHIA COLI.	E coli Proteins
D035001	Host Factor 1 Protein	An integration host factor that was originally identified as a bacterial protein required for the integration of bacteriophage Q beta (ALLOLEVIVIRUS). Its cellular function may be to regulate mRNA stability and processing in that it binds tightly to poly(A) RNA and interferes with ribosome binding.	Host Factor 1,Host Factor Q,hfq Gene Product