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 fluor escence-detected temperature jump relaxation. The ribozyme was substituted at different positions by 2-aminopurine (2-AP) as fluorescence indicator; t hese substitutions do not:inhibit catalysis. The general shape of relaxatio n curves reported from different positions of the ribozyme is very similar: a fast decrease of fluorescence,mainly due to physical quenching, is follo wed by a slower increase of fluorescence due to conformational relaxation. In most cases at least three-relaxation time constants in the time range fr om a few microseconds to similar to 200 ms are required for fitting. Althou gh the relaxation at different positions of the ribozyme is similar in gene ral, suggesting a global type of ribozyme dynamics, a close examination rev eals differences, indicating an individual local response. For:example, 2-A P 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, r esulting in an inactive form, leads to the appearance of a particularly slo w relaxation process (tau approximate to 200 ms). addition of Mg2+ ions ind uces a reduction of amplitudes and in most cases a general increase of time constants. Differences between the hammerhead variants are clearly demonst rated by subtraction of relaxation:curves recorded under corresponding cond itions. The changes induced in the relaxation response by Mg2+ are very sim ilar to those induced by Ca2+. The relaxation data do not provide any evide nce for formation of Mg2+-inner sphere complexes in hammerhead ribozymes, b ecause 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 co mplexation. Amplitudes and time constants show that the equilibrium constan t of inner sphere complexation is 1.2, corresponding to 55% inner sphere st ate of the Mg2+ complexes; the rate constant 6.6 x 10(3) s(-1) for inner sp here complexation is relatively low and shows the existence of some barrier (s) on the way to inner sphere complexes.