A Three-Dimensional Model for the Hammerhead Ribozyme Based on Fluorescence Measurements

Abstract

For the understanding of the catalytic function of the RNA hammerhead ribozyme, a three-dimensional model is essential but neither a crystal nor a solution structure has been available. Fluorescence resonance energy transfer (FRET) was used to study the structure of the ribozyme in solution in order to establish the relative spatial orientation of the three constituent Watson-Crick base-paired helical segments. Synthetic constructs were labeled with the fluorescence donor (5-carboxyfluorescein) and acceptor (5-carboxytetramethylrhodamine) located at the ends of the strands constituting the ribozyme molecule. The acceptor helix in helix pairs I and III and in II and III was varied in length from 5 to 11 and 5 to 9 base pairs, respectively, and the FRET efficiencies were determined and correlated with a reference set of labeled RNA duplexes. The FRET efficiencies were predicted on the basis of vector algebra analysis, as a function of the relative helical orientations in the ribozyme constructs, and compared with experimental values. The data were consistent with a Y-shaped arrangement of the ribozyme with helices I and II in close proximity and helix III pointing away. These orientational constraints were used for molecular modeling of a three-dimensional structure of the complete ribozyme.

Diffusely Bound Mg2+ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

“…In previous measurements with HHR at pH 6, an even higher apparent Mg 2+ dissociation constant was found than in our measurements at pH 8, indicating a possible pH dependence of Mg 2+ binding (24). Steady-state fluorescence spectroscopy has been previously used to determine the mean FRET efficiency of fluorophorelabeled hammerhead ribozymes (7,8). These steady-state experiments, however, can only provide qualitative information about the fluorophore distances, whereas a technique such as time-resolved FRET (tr-FRET) yields quantitative distances between the donor and acceptor fluorophores (28,30).…”

“…Earlier solution studies of the hammerhead ribozyme have used steady-state FRET to measure the efficiency of a single energy transfer between two fluorophores attached to stems I and II (7,8,29). This energy transfer efficiency is related to the mean stem I-stem II distance derived from our tr-FRET measurements.…”

Computed Energetics of Nucleotides in Spatial Ribozyme Structures: An Accurate Identification of Functional Regions from Structure

“…These calculations were performed using a "multiple trajectories" technique [26,27] that is more likely to produce models closer to experimental structures than simply an energy minimization or a singular long trajectory of molecular dynamics. Apart from NMR and crystal structures of ribozymes [1,6,7,8,9,10,11,12,13,28,29,30,31,32], the method was also applied to RNAs of other types [33,34,35] as well as to several structural models of ribozymes [36,37,38].…”

“…For example, there is a model of hammerhead ribozyme based primarily on fluorescence measurements [36]. The structure itself does not provide any indications where the active site might be located [36].…”

“…For example, there is a model of hammerhead ribozyme based primarily on fluorescence measurements [36]. The structure itself does not provide any indications where the active site might be located [36]. However, the structural and biochemical data on other hammerhead ribozymes (1hmhA and 300dA in Tables 1 and 2) suggest that the catalytic residues in this hammerhead model would be among C6 , G8, A9, G11, G13, A28, U43, and C44.…”