EPR Spectroscopic Analysis of U7 Hammerhead Ribozyme Dynamics during Metal Ion Induced Folding
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- 作者：Thomas E. Edwards and Snorri Th. Sigurdsson
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：September 27, 2005
- 年：2005
- 卷：44
- 期：38
- 页码：12870 - 12878
- 全文大小：127K
- 年卷期：v.44,no.38(September 27, 2005)
- ISSN：1520-4995
文摘
Electron paramagnetic resonance (EPR) spectroscopy was used to examine changes in internalstructure and dynamics of the hammerhead ribozyme upon metal ion induced folding, changes in pH, andthe presence and absence of ribozyme inhibitors. A nitroxide spin-label was attached to nucleotide U7 ofthe HH16 catalytic core, and this modified ribozyme was observed to retain catalytic activity. U7 wasshown by EPR spectroscopy to be more mobile in the ribozyme-product complex than in either theunfolded ribozyme or the ribozyme-substrate complex. A two-step divalent metal ion dependent foldingpathway was observed for the ribozyme-substrate complex with a weak first transition observed at 0.25mM Mg2+ and a strong second transition observed around 10 mM Mg2+, in agreement with studies usingother biophysical and biochemical techniques. Previously, ribozyme activity was observed in the absenceof divalent metal ions and the presence of high concentrations of monovalent metal ions, although theactivity was less than that observed in the presence of divalent metal ions. Here, we observed similardynamics for U7 in the presence of 4 M Na+ or Li+, which were distinctively different than that observedin the presence of 10 mM Mg2+, indicating that U7 of the catalytic core forms a different microenvironmentunder 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 onedivalent 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 RNAfolding and function.