Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy

N Kisseleva, A Khvorova, E Westhof, Olav Schiemann

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52 Citations (Scopus)

Abstract

Electron paramagnetic resonance (EPR) spectroscopy is used to study the binding of Mn-II ions to a tertiary stabilized hammerhead ribozyme (tsHHRz) and to compare it with the binding to the minimal hammerhead ribozyme (mHHRz). Continuous wave EPR measurements show that the tsHHRz possesses a single high-affinity Mn-II binding site with a K-D of less than or equal to10 nM at an NaCl concentration of 0.1 M. This dissociation constant is at least two orders of magnitude smaller than the K, determined previously for the single high-affinity Mn-II site in the mHHRz. In addition, whereas the high-affinity Mn-II is displaced from the mHHRz upon binding of the aminoglycoside antibiotic neomycin B, it is not from the tsHHRz. Despite these pronounced differences in binding, a comparison between the electron spin echo envelope modulation and hyperfine sublevel correlation spectra of the minimal and tertiary stabilized HHRz demonstrates that the structure of both binding sites is very similar. This suggests that the Mn-II is located in both ribozymes between the bases A9 and G10.1 of the sheared G (.) A tandem base pair, as shown previously and in detail for the mHHRz. Thus, the much stronger Mn-II binding in the tsHHRz is attributed to the interaction between the two external loops, which locks in the RNA fold, trapping the Mn-II in the tightly bound conformation, whereas the absence of long-range loop-loop interactions in the mHHRz leads to more dynamical and open conformations, decreasing Mn-II binding.

Original languageEnglish
Pages (from-to)1-6
Number of pages6
JournalRNA
Volume11
Issue number1
DOIs
Publication statusPublished - Jan 2005

Keywords

  • hammerhead ribozyme
  • EPR
  • manganese
  • antibiotic
  • RNA
  • ESEEM
  • METAL-ION BINDING
  • CLEAVAGE ACTIVITY
  • EPR SPECTROSCOPY
  • MN2+ SITES
  • ELEMENTS
  • SPECTRA
  • PELDOR
  • BAND

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