Biochemical detection of monovalent metal ion binding sites within RNA.
Many RNAs, including the ribosome, RNase P, and the group II intron, explicitly require monovalent cations for activity in vitro. Although the necessity of monovalent cations for RNA function has been known for more than a quarter of a century, the characterization of specific monovalent metal sites within large RNAs has been elusive. Here we describe a biochemical approach to identify functionally important monovalent cations in nucleic acids. This method uses thallium (Tl+), a soft Lewis acid heavy metal cation with chemical properties similar to those of the physiological alkaline earth metal potassium (K+). Nucleotide analog interference mapping (NAIM) with the sulfur-substituted nucleotide 6-thioguanosine in combination with selective metal rescue of the interference with Tl+ provides a distinct biochemical signature for monovalent metal ion binding. This approach has identified a K+ binding site within the P4-P6 domain of the Tetrahymena group I intron that is also present within the X-ray crystal structure. The technique also predicted a similar binding site within the Azoarcus group I intron where the structure is not known. The approach is applicable to any RNA molecule that can be transcribed in vitro and whose function can be assayed.
Basu
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Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven, Connecticut 06520-8114, USA.
Strobel
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D001665:Binding Sites; D002412:Cations / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D007477:Ions; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D013872:Thionucleosides / Q000378:metabolism
D002412:Cations; D007477:Ions; D008670:Metals; D013872:Thionucleosides; D006151:Guanosine; D012313:RNA; C005783:6-thioguanosine