Nucleic Acids Research, 2000, Vol. 28, No. 22 4428-4434
© 2000 Oxford University Press
Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics
Max Planck Institut für biophysikalische Chemie, D-37077 Göttingen, Germany and 1Max Planck Institut für experimentelle Medizin, D-37075 Göttingen, Germany
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 ~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 (
200 ms). Addition of Mg2+ 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 Mg2+ are very similar to those induced by Ca2+. The relaxation data do not provide any evidence for formation of Mg2+-inner sphere complexes in hammerhead ribozymes, because a Mg2+-specific relaxation effect was not visible. However, a Mg2+-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 Mg2+ complexes; the rate constant 6.6 x 103 s–1 for inner sphere complexation is relatively low and shows the existence of some barrier(s) on the way to inner sphere complexes.
* To whom correspondence should be addressed. Tel: +49 551 2011438; Fax: +49 551 2011168; Email: dpoersc{at}gwdg.de
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