Mn2+-Nitrogen Interactions in RNA Probed by Electron Spin-Echo Envelope Modulation Spectroscopy: Application to the Hammerhead Ribozyme

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• 作者：Susan R. Morrissey ; Thomas E. Horton ; Christopher V. Grant ; Charles G. Hoogstraten ; R. David Britt ; and Victoria J. DeRose
• 刊名：Journal of the American Chemical Society
• 出版年：1999
• 出版时间：October 6, 1999
• 年：1999
• 卷：121
• 期：39
• 页码：9215 - 9218
• 全文大小：49K
• 年卷期：v.121,no.39(October 6, 1999)
• ISSN：1520-5126

文摘

We report application of electron spin-echo envelope modulation (ESEEM) spectroscopy to theproblem of metal coordination environments in structured RNA molecules. ESEEM has been used in conjunctionwith ¹⁵N-guanosine labeling to identify nitrogen ligation to a Mn²⁺ site in a hammerhead ribozyme and inMn²⁺-model guanosine monophosphate (GMP) complexes. Hammerhead ribozyme complexes consisting ofa 34-nucleotide RNA enzyme strand annealed to a 13-nucleotide DNA substrate strand were poised in 1 MNaCl as a 1:1 complex with Mn²⁺, conditions previously determined to populate a single high-affinity Mn²⁺site (Horton, T. E.; Clardy, R. D.; DeRose, V. J. Biochemistry 1998, 51, 18094-18108). Significant modulationof the electron spin-echo from several low-frequency features is detected for the natural-abundance, ¹⁴N-hammerhead samples. At 3600 G, the main hammerhead three-pulse ESEEM features arise at 0.6, 1.9, 2.5,and 5.2 MHz and are nearly identical for a Mn²⁺-GMP complex under the same conditions. For a ribozymehaving ¹⁵N-guanosine incorporated into the enzyme strand, as well as for an ¹⁵N-labeled Mn²⁺-GMP complex,the modulation is completely altered and consists of one main feature at 3.4 MHz and a smaller feature at themages/gifchars/nu.gif" BORDER=0 >_n(¹⁵N) Larmor frequency of 1.6 MHz. Preliminary analysis of the ESEEM data reveals an apparent hyperfinecoupling of A(¹⁴N) ~ 2.3 MHz, similar to previously reported values for Mn²⁺ directly coordinated to histidineand imidazole. These data demonstrate the potential for ESEEM as a spectroscopic tool for metal liganddetermination in structured RNA molecules.