Ribozymes interactions between ribozyme, substrates

Stuhlmann, F., Jaschke, A. Chai acterization of an RNA active site interactions between a Diels-Alderase ribozyme and its substrates and products. J. Am. Chem. Soc. 124, 3238-3244 (2002). 

By analogy with protein enzymes and precepts of transition state theory, ribozyme catalysis should involve an array of interactions between RNA functional groups and the reacting groups of the substrate that lower the free energy of the... 

Some aptamer beacons using interactions between fluorescence and quenchers have been reviewed by Jayasena, 111). On the other hand, Hartig et al. 112) developed a ribozyme-based detection scheme where an external substrate labeled with a fluorescent molecule at its 5 end and a fluorescence quencher at its 3 end was cleaved, resulting in a fluorescence dequenching, enabling real-time monitoring of ribozyme activity. 

Figure 1. Catalysis and template action of RNA and proteins. Catalytic action of one RNA molecule on another one is shown in the simplest case, the "hammerhead ribozyme." The substrate is a tridecanucleotide forming two double-helical stacks together with the ribozyme (n = 34) in the confolded complex. Tertiary interactions determine the detailed structure of the hammerhead ribozyme complex and are important for the enzymatic reaction cleaving one of the two linkages between the two stacks. Substrate specificity of ribozyme catalysis is caused by secondary structure in the cofolded complex between substrate and catalyst. Autocatalytic replication of oligonucleotide and nucleic acid is based on G = C and A = U complementarity in the hydrogen bonded complexes of nucleotides forming a Watson-Crick type double helix. Gunter von Kiedrowski s experi-...

For combination in a twin ribozyme both units should specifically interact with a unique substrate sequence. To this end we changed the substrate sequence in the single units and determined the kinetic constants of the cleavage reaction before constructing the twin ribozymes. All single catalytic modules under single turnover conditions had rate constants between 0.2 and 0.5 min-1. 

The Gl ribozyme reaction depends on the presence of divalent metal ions but as indicated above, the binding of these ions plays multiple roles that include folding and enhancing substrate binding affinities (59). The rate of the chemical step is Mg(2- -) dependent, but these data do not distinguish between direct or indirect roles, or a combination of both. As indicated above, distinguishing active site metal ions from what has been referred to as the sea of other functionally important metal ion interactions presents a considerable challenge. For the GI ribozyme and other catalytic RNAs, site-specific evidence for active site metal interactions comes primarily from analyses of thiophilic metal ion rescue of phosphorothioate and other substrate modifications (e.g.. References 60 and 61). These analyses rely on the fact that substitution of a substrate phosphate by a phosphorothioate weakens the affinity of coordinated Mg(2- -) ions... 

An RNA Diels-Alderase ribozyme recently developed that catalyses the formation of carbon-carbon bonds between a tethered anthracene diene and a biotinylated maleimide dienophile. The ribozyme active site has been further characterised by chemical substitution of the diene and dienophile. It was shown that the diene must be an anthracene, and substitution only at specific sites is permitted. The dienophile must be a maleimide with an unsubstituted double bond. The RNA-diene interaction was found to be governed preferentially by stacking interactions. A ribozyme has been selected that catalyses the synthesis of dipeptides using an aminoacyl-adenylate substrate. The ribozyme catalysed the formation of 30 different dipeptides, many with rates similar to that of the Met-Phe dipeptide used in the selection process. 

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