Ribozyme-substrate complex

Hampel and Burke observed that protection of hammerhead backbone sites in Mg + solutions required assembly of the full ribozyme-substrate complex. In other words, testing of ribozyme or substrate separately in the hydroxyl footprinting assay showed essentially complete hydrolysis of all nucleotides (Figure 2B of reference 56). In contrast, the fully assembled ribozyme-substrate complex showed protection of nucleotides structurally near the densely packed three-helix junction of hammerhead constructs HH16, HHal, and RNA 6. Two of the ribozyme group of protected nucleotides (Gs, Ae) are part of the conserved uridine U-turn seen in all known hammerhead constructs. (See Figures 6.10,6.11, and 6.12.) The footprinting results are collected in Table 6.5. 

It is generally accepted that the tertiary structures of RNA molecules are stabihzed by metal ions. The roles of metal ions in ribozyme-catalyzed reactions fall into two distinct types the metal ions can act as catalysts during the chemical cleavage step, as shown in Fig. 3, and they can also stabilize the conformation of the ribozyme-substrate complex. 

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... 

This inhibitory effect of NaCl can be explained on the basis of the data from Horton et al. [81] that is described above. The Na ions remove Mn ions from the lower affinity site(s), which is somehow involved in the ribozyme activity, from the ribozyme-substrate complex. When Hammann et al. used ions in their NMR analysis, they noticed that the apparent Ka for the lower affinity ions depended on the concentration of Na ions [80]. An increase in the background concentration of NaCl from 10 mmol/1 to 50 mmol/1 weakened the affinity of the Mg ions for the complex. Their observations also reconcile with the observed inhibition by Na ions in our study, with the competitive removal of Mg /Mn ions from the ribozyme-substrate complex. 

Zaug, A. J., Grosshans, C. A., and Cech, T. R. (1988). Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes. Biochemistry 27, 8924-8931. 

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. 

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