EPR spectroscopic analysis of U7 hammerhead ribozyme dynamics during metal ion induced folding.
Electron paramagnetic resonance (EPR) spectroscopy was used to examine changes in internal structure and dynamics of the hammerhead ribozyme upon metal ion induced folding, changes in pH, and the presence and absence of ribozyme inhibitors. A nitroxide spin-label was attached to nucleotide U7 of the HH16 catalytic core, and this modified ribozyme was observed to retain catalytic activity. U7 was shown by EPR spectroscopy to be more mobile in the ribozyme-product complex than in either the unfolded ribozyme or the ribozyme-substrate complex. A two-step divalent metal ion dependent folding pathway was observed for the ribozyme-substrate complex with a weak first transition observed at 0.25 mM Mg2+ and a strong second transition observed around 10 mM Mg2+, in agreement with studies using other biophysical and biochemical techniques. Previously, ribozyme activity was observed in the absence of divalent metal ions and the presence of high concentrations of monovalent metal ions, although the activity was less than that observed in the presence of divalent metal ions. Here, we observed similar dynamics for U7 in the presence of 4 M Na+ or Li+, which were distinctively different than that observed in the presence of 10 mM Mg2+, indicating that U7 of the catalytic core forms a different microenvironment under monovalent versus divalent metal ion conditions. Interestingly, the catalytically efficient microenvironment of U7 was similar to that observed in a solution containing 1 M Na+ upon addition of one divalent metal ion per ribozyme. In summary, these results demonstrate that changes in local dynamics, as detected by EPR spectroscopy, can be used to study conformational changes associated with RNA folding and function.
Edwards
Thomas E
TE
Department of Chemistry, University of Washington, Seattle, Washington 98195-1700, USA.
Sigurdsson
Snorri Th
ST
D002413:Cations, Divalent / Q000737:chemistry; D002414:Cations, Monovalent / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D009589:Nitrogen Oxides / Q000737:chemistry; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D012964:Sodium / Q000737:chemistry; D013113:Spin Labels
D002413:Cations, Divalent; D002414:Cations, Monovalent; D004791:Enzyme Inhibitors; D008670:Metals; D009589:Nitrogen Oxides; D016337:RNA, Catalytic; D013113:Spin Labels; C427204:hammerhead ribozyme; D008094:Lithium; D012964:Sodium; D008274:Magnesium