Ribozyme-substrate complex, folding

The folding pathway of the ribozyme-substrate complex upon addition of metal ions is also well studied. Bassi et al. analyzed the global structure of... 

Fig. 7 The two-stage folding scheme for the hammerhead ribozyme, as proposed by Tilley s group [77-80]. The arrow indicates the cleavage site. The scheme consists of two steps to generate the Y- or y-shaped ribozyme/substrate complex. The higher affinity of Mg is related to formation of domain II (structural scaffold non-Watson-Crick pairings between G12-A9, Ais-Gg and A14-U7 forming a coaxial stack between hehces II and III that runs through G12A13A14) and the lower affinity of Mg to formation of domain I (catalytic domain formation by the sequence C3U4G5A6 and the C17 with the rotation of helix I around into the same quadrant as helix II) [78]...

Horton et al. analyzed the Mn -binding properties of hammerhead ribozyme-substrate complexes by EPR [81]. The results are consistent with the two-phase folding model. They found two classes of metal-binding sites with higher affinity and lower affinity, by monitoring the number of bound Mn + ions per hammerhead ribozyme-substrate complex at various concentrations of NaCl. They observed, in the presence of a constant concentration of Mn + ions, a sudden decrease in the number of bound low-affinity Mn ions at a lower concentration of NaCl, followed by a slow decrease or a plateau value of the number of bound high-affinity Mn ions at a higher concentration of NaCl. For example, in the absence of NaCl and in the presence of either 0.3 mmol/1 or 1 mmol/1 Mn + ions, the number of bound Mn ions per hammerhead ribozyme-substrate complex was approximately 14. Addition... 

Two independently-folding domains. A variety of methods were applied to examine the structure of intact hairpin ribozymes, ribozyme-substrate complexes, and ribozyme derivatives in which some sequences were deleted. Crosslinking studies, gel mobility analysis, and chemical modification experiments suggested that the ribozyme consists of two independently-folding domains. Domain A is a duplex between the substrate and substrate-binding strand (helix 1, loop A and helix 2), while domain B consists of helix 3, loop B and helix 4. 

In our recent study of reaction kinetics, we observed an unusual phenomenon when we analyzed the activity of a hammerhead ribozyme as a function of the concentration of Na ions on a background of a low concentration of either Mn or Mg ions [82]. At lower concentrations of Na ions, Na ions had an inhibitory effect on ribozyme activity, whereas at higher concentrations, Na ions had a rescue effect. We propose that these observations can be explained if we accept the existence of two kinds of metal-binding site that have different affinities. Our data also support the two-phase folding theory [77-80, Fig. 7], in which divalent metal ions in the ri-bozyme-substrate complex have lower and higher affinities, as proposed by Lilley and coworkers on the basis of their observations of ribozyme complexes in the ground state. 

Hydrolytic catalysis by metal ions is also important in the hydrolysis of nucleic acids, especially RNA (36). Molecules of RNA that catalyze hydrolytic reactions, termed ribozymes, require divalent metal ions to effect hydrolysis efficiently. Thus, all ribozymes are metalloenzymes (6). There is speculation that ribozymes may have been the first enzymes to evolve (37), so the very first enzymes may have been metalloenzymes Recently, substitution of sulfur for the 3 -oxygen atom in a substrate of the tetrahymena ribozyme has been shown to give a 1000-fold reduction in rate of hydrolysis with Mg2+ but no attenuation of the hydrolysis rate with Mn2+ and Zn2+ (38). Because Mn2+ and Zn2+ have stronger affinities for sulfur than Mg2+ has, this feature provides strong evidence for a true catalytic role of the divalent cation in the hydrolytic mechanism, involving coordination of the metal to the 3 -oxygen atom. Other examples of metal-ion catalyzed hydrolysis of RNA involve lanthanide complexes, which are discussed in this volume. 

Figure 2.5. Catalytic RNA. (A) The base-pairing pattern of a "hammerhead" ribozyme and its substrate. (B) The folded conformation of the complex. The ribozyme cleaves the bond at the cleavage site. The paths of the nucleic acid backbones are highlighted in red and blue. 

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