The different role of high- and low-affinity metal ions in cleavage by a tertiary stabilized cis hammerhead ribozyme from tobacco ringspot virus

Natalia Kisseleva, Anastasia Khvorova, Eric Westhof, Olav Schiemann, Alexey D. Wolfson*

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

Abstract

The aim of this study was to investigate the dependence of the observed cleavage rates (k(obs)) of a tertiary stabilized hammerhead ribozyme (tsHHRz) and of a minimal hammerhead ribozyme (mHHRz), both derived from tobacco ringspot virus, on the type and concentration of divalent metal ions in order to interpret the functional role of high-affinity ions detected by electron paramagnetic resonance (EPR). To measure the fast cleavage of the cis tsHHRz, a new method using chemically synthesized fluorescent-labeled RNAs has been developed. The tsHHRz cleavage rate is up to 20-fold faster than that of the mHHRz under similar conditions. The presence of Mn2+ ions leads to a 60-fold faster cleavage than in the presence of Mg2+ ions. The functional role of the high-affinity ion was evaluated using neomycin B inhibition studies. Neomycin B reduces the cleavage activity of both ribozymes but the inhibitory effect on tsHHRz is much weaker than that on the mHHRz. EPR data had shown that neomycin B displaces both low-affinity and high-affinity Mn2+ ions from the mHHRz, but only low-affinity ions from tsHHRz. Inhibition of the tsHHRz activity may be due to the displacement of weakly bound Me2+ ions required for the local folding leading to cleavage, whereas both the high-affinity ion required for folding and the weakly bound ions are replaced in the mHHRz. The high-affinity metal ion is required for the stabilization of the global HHRz structure, but is not involved in catalysis or stabilization of the transient state.

Original languageEnglish
Pages (from-to)101-110
Number of pages10
JournalOligonucleotides
Volume18
Issue number2
DOIs
Publication statusPublished - Jun 2008

Keywords

  • SELF-CLEAVAGE
  • MONOVALENT CATIONS
  • NUCLEIC-ACIDS
  • BINDING
  • RNA
  • NEOMYCIN
  • REQUIREMENTS
  • SPECTROSCOPY
  • INHIBITION
  • MECHANISM

Fingerprint

Dive into the research topics of 'The different role of high- and low-affinity metal ions in cleavage by a tertiary stabilized cis hammerhead ribozyme from tobacco ringspot virus'. Together they form a unique fingerprint.

Cite this