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

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 similar 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 dynamjcs. 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 approximate to 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 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.