Biomaterial Database

RNA catalytic activity as a probe of chaperone-mediated RNA folding. 2014

Brant Gracia, and Rick Russell

Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.

For structured RNAs that possess catalytic activity, this activity provides a powerful probe for measuring the progress of folding and the effects of RNA chaperone proteins on the folding rate. The crux of this approach is that only the natively folded RNA is able to perform the catalytic reaction. This method can provide a quantitative measure of the fraction of native RNA over time, and it can readily distinguish the native state from all misfolded conformations. Here we describe an activity-based method measuring native folding of ribozymes derived from self-splicing group I introns, and we show how the assay can be used to monitor acceleration of native folding by DEAD-box RNA helicase proteins that function as general RNA chaperones. By measuring the amount of substrate that is converted to product in a rapid first turnover, we describe how to determine the fraction of the ribozyme population that is present in the native state. Further, we describe how to perform a two-stage or discontinuous assay in which folding proceeds in stage one and then solution conditions are changed in stage two to permit catalytic activity and block further folding. This protocol allows folding to be followed under a broad range of solution conditions, including those that do not support catalytic activity, and facilitates studies of chaperone proteins.

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
D004591	Electrophoresis, Polyacrylamide Gel	Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.	Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D001345	Autoradiography	The making of a radiograph of an object or tissue by recording on a photographic plate the radiation emitted by radioactive material within the object. (Dorland, 27th ed)	Radioautography
D013768	Tetrahymena	A genus of ciliate protozoa commonly used in genetic, cytological, and other research.	Tetrahymenas
D016337	RNA, Catalytic	RNA that has catalytic activity. The catalytic RNA sequence folds to form a complex surface that can function as an enzyme in reactions with itself and other molecules. It may function even in the absence of protein. There are numerous examples of RNA species that are acted upon by catalytic RNA, however the scope of this enzyme class is not limited to a particular type of substrate.	Catalytic RNA,Ribozyme,Ribozymes
D053487	DEAD-box RNA Helicases	A large family of RNA helicases that share a common protein motif with the single letter amino acid sequence D-E-A-D (Asp-Glu-Ala-Asp). In addition to RNA helicase activity, members of the DEAD-box family participate in other aspects of RNA metabolism and regulation of RNA function.	DEAD-box RNA Helicase,DEAD Box Helicase p68,p68 DEAD Box Protein,p68 RNA Helicase,DEAD box RNA Helicase,DEAD box RNA Helicases,Helicase, DEAD-box RNA,Helicase, p68 RNA,Helicases, DEAD-box RNA,RNA Helicase, DEAD-box,RNA Helicase, p68,RNA Helicases, DEAD-box
D059370	RNA Folding	The processes of RNA tertiary structure formation.	RNA Refolding,RNA Unfolding,Folding, RNA,Foldings, RNA,RNA Foldings,RNA Refoldings,RNA Unfoldings,Refolding, RNA,Refoldings, RNA,Unfolding, RNA,Unfoldings, RNA