Global Structure and Flexibility of Hairpin Ribozymes with Extended Terminal Helices

Abstract

Global structure and flexibility of three different hairpin ribozyme constructs have been analyzed by measuring their electric dichroism decay in various buffers at temperatures between 2 and 30°C. The hairpin ribozyme is characterized by two independently folding domains A and B that are connected through a hinge and have to interact to enable catalysis. The analyzed constructs feature extended terminal helices 1 and 4 with 27 and 25bp, respectively, to increase the sensitivity of the molecular rotational diffusion time constants with respect to the interdomain bending angle. Constructs HP1 and HP2 cannot cleave because of a G+1A change at the 3′-side of the cleavage site; in HP1 the helices 2 and 3 that flank the hinge form a continuous double helical segment; in HP2 and HP3, a six nucleotide bulge confers flexibility to the expected bending site; HP3 is a cleavable form of HP2 with a G+1-base. For comparison, a standard RNA double helix with 72bp was included in our analysis. The dichroism decay curves of the hairpin constructs after pulses of low electric field strengths can be fitted to single exponentials τs, whereas the curves after pulses of high field strengths require two exponentials. In all cases, time constants increase with RNA concentration, indicating intermolecular interactions. Extrapolation of the τsvalues measured in standard buffer (50mM Tris (pH 7.5) and 12mM MgCl2) to zero RNA concentration provide values of 112, 93, and 73 ns for HP1, HP2 and HP3, respectively, at 30°C, indicating increasingly compact structures. The 72bp RNA reference under corresponding conditions did not show a dependence of its decay time constant on the RNA concentration nor on the field strength; its time constant is 175 ns (standard buffer, 30°C). The observation of two relaxation processes for the hairpin constructs at high field strengths indicates stretching to a more elongated state; the fast process with a time constant of the order of 50 ns is assigned to reversion of stretching, the slow process to overall rotation. The overall rotational time of the stretched state at 20°C is close to that for a completely stretched rigid state; at 30°C the experimental values are around 70% of that expected for a completely stretched rigid state, indicating flexibility and/or residual bending. Bead models were constructed to simulate dichroism decay curves. The time constants observed for the 72bp RNA are as expected for a rigid rod with a rise of 2.8Å per base-pair. Based on this rise per base-pair for models of a V and a Y-shape, we estimate average bending angles of 80(±20)° and 105 (±25)°, respectively, for the catalytically active hairpin ribozyme HP3. The energy required for stretching is of the order of the thermal energy.