NnnnnnnnnwN Fig3i ATPnd

(j the corresponding aptamer

Fig. 3.9 Self-incorporation of coenzyme (adapted from Breaker and Joyce, 1995).

mmol/L). This is of particular interest as two messenger RNA riboswitches from Escherichia coli are known to discriminate between phosphorylated small molecules.

The first self-incorporation of a coenzyme into a ribozyme was performed in 1995 by Breaker and Joyce (1995), substituting neatly alternative coenzymes into the primary structure of group I intron by replacing the guanosine substrate with natural coenzymes or analogs (Fig. 3.9a and b respectively).

Looking for significant metabolic reactions and for coenzyme-dependent ribo-zymes, the primary biological cofactor used in acyltransfer reactions, coenzyme A (CoA), has been the target of RNA pools leading to a 52-nucleotide minimal aptamer (Burke and Hoffmann, 1998) which recognizes the adenosine moiety of CoA and binds others ATP analogs (Fig. 3.10). The selection of coenzyme synthetase ribozymes is of particular interest in an RNA-catalyzed energy metabolism. Yarus, exploring the origin of ribonucleotide coenzymes, demonstrated the RNA-catalyzed formation of three common coenzymes CoA, NAD, and FAD from their precursors, 4'-phosphopantetheine, NMN, and FMN, respectively (Huang et al., 2000). A ribozyme capable of utilizing CoA for the synthesis of acyl-CoA was selected in vitro. The co-ribozyme isolated, that is an acyl-CoA synthetase, produced acetyl-CoA and butyryl-CoA (Jadhav and Yarus, 2002b).

The use of organic cofactors has been illustrated by a hairpin ribozyme that requires adenine as a cofactor during RNA reversible self-cleavage reaction (Meli et al., 2003). This may lead to a better understanding of prebiotic cofactors in primeval catalysis. Our working hypothesis is based on the demonstration of

Secondary structures of coenzyme-RNA aptamers.

Secondary structures of coenzyme-RNA aptamers.

esterase activity in a nucleoside analog, the N6-ribosyladenine (Fuller et al., 1972; Maurel and Ninio, 1987). The activity, due to the presence of an imidazole group that is free and available for catalysis, is comparable to that of histidine placed in the same conditions (Fig. 3.11). We have studied the kinetic behavior of this type of catalyst (Ricard et al., 1996) and have shown that the catalytic effect increases greatly when the catalytic element, the pseudohistidine, is placed in a favorable environment within a macromolecule (D├ęcout et al., 1995). Moreover, primitive nucleotides were not necessarily restricted to the standard nucleotides encountered today, and because of their replicative and catalytic properties, the N6 and N3 substituted derivatives of purines could have constituted essential links between the nucleic acid world and the protein world.

Following this line of investigation we started the selection of ribozymes dependent on adenine. Starting from a heterogeneous population of RNAs with 1015 variants (a population of 1015 different molecules) we have selected five populations of RNAs capable of specifically recognizing adenine after ten generations (Meli et al., 2002). When cloned, sequenced, and modeled, the best one among the individuals of these populations, has a shape reminiscent of a claw capable of grasping adenine. Following this result we have isolated from a degenerated hairpin ribozyme, by in vitro selection, two adenine-dependent ribozymes capable of triggering reversible cleavage reactions (Fig. 3.12). One of them is also active with imidazole alone (Meli et al., 2003).

A quarter of classified enzymatic reactions are redox reactions involved in various biological events, such as metabolism of biological molecules, detoxification, energy production, and regulation of protein functions. An RNA molecule binding hemin and exhibiting a peroxidase activity has been reported (Travascio et al., 1999). In this case, RNA and DNA of the same nucleotide sequence are capable of forming comparable cofactor-binding sites promoting catalysis.

Fig. 3.11 (a) Adenine. (b) Comparison of modified adenosine and histidine. (c) Catalytic activity of adenine residue.

Was this article helpful?

0 0
Turbo Metabolism

Turbo Metabolism

Forget Silly Diets-They Don't Work. Weight loss has got to be the most frustrating experience for many people, young and old alike. Eating foods that are just horrible, denying yourself foods you truly love and enjoy. Exercising, even though you absolutely hate exercising, and end up stiff as a board with no results.

Get My Free Ebook

Post a comment