Help Privacy Policy Disclaimer
  Advanced SearchBrowse


  Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics

Menger, M., Eckstein, F., & Poerschke, D. (2000). Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics. Nucleic Acids Research, 28, 4428-4434.

Item is


show Files




Menger, M.1, Author           
Eckstein, F., Author
Poerschke, D.2, Author
1Abteilung Biochemische Kinetik, MPI for biophysical chemistry, Max Planck Society, ou_578616              
2Max Planck Society, ou_persistent13              


Free keywords: none available
 Abstract: The dynamics of a hammerhead ribozyme was analyzed by measurements of fluorescence-detected temperature jump relaxation. The ribozyme was substituted at different positions by 2-aminopurine (2-AP) as fluorescence indicator; these substitutions do not inhibit catalysis. The general shape of relaxation curves reported from different positions of the ribozyme is very similar: a fast decrease of fluorescence, mainly due to physical quenching, is followed by a slower increase of fluorescence due to conformational relaxation. In most cases at least three relaxation time constants in the time range from a few microseconds to approximately 200 ms are required for fitting. Although the relaxation at different positions of the ribozyme is similar in general, suggesting a global type of ribozyme dynamics, a close examination reveals differences, indicating an individual local response. For example, 2-AP in a tetraloop reports mainly the local loop dynamics known from isolated loops, whereas 2-AP located at the core, e.g. at the cleavage site or its vicinity, also reports relatively large amplitudes of slower components of the ribozyme dynamics. A variant with an A-->G substitution in domain II, resulting in an inactive form, leads to the appearance of a particularly slow relaxation process (tau approximately 200 ms). Addition of Mg(2+) ions induces a reduction of amplitudes and in most cases a general increase of time constants. Differences between the hammerhead variants are clearly demonstrated by subtraction of relaxation curves recorded under corresponding conditions. The changes induced in the relaxation response by Mg(2+) are very similar to those induced by Ca(2+). The relaxation data do not provide any evidence for formation of Mg(2+)- inner sphere complexes in hammerhead ribozymes, because a Mg(2+)- specific relaxation effect was not visible. However, a Mg(2+)-specific effect was found for a dodeca-riboadenylate substituted with 2-AP, showing that the fluorescence of 2-AP is able to indicate inner sphere complexation. Amplitudes and time constants show that the equilibrium constant of inner sphere complexation is 1.2, corresponding to 55% inner sphere state of the Mg(2+) complexes; the rate constant 6.6 x 10(3) s(-1) for inner sphere complexation is relatively low and shows the existence of some barrier(s) on the way to inner sphere complexes.


Language(s): eng - English
 Dates: 2005-08-172000
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 231149
Other: 23137
 Degree: -



Legal Case


Project information


Source 1

Title: Nucleic Acids Research
Source Genre: Journal
Publ. Info: -
Pages: - Volume / Issue: 28 Sequence Number: - Start / End Page: 4428 - 4434 Identifier: -