Adenine-dependent hairpin ribozymes were isolated by in vitro selection from a degenerated hairpin ribozyme population. Two new adenine-dependent ribozymes catalyze their own reversible cleavage in the presence of free adenine. Both aptamers have Mg 2؉ requirements for adenine-assisted cleavage similar to the wild-type hairpin ribozyme. Cleavage kinetics studies in the presence of various other small molecules were compared. The data suggest that adenine does not induce RNA self-cleavage in the same manner for both aptamers. In addition, investigations of pH effects on catalytic rates show that both adenine-dependent aptamers are more active in basic conditions, suggesting that they use new acid/base catalytic strategies in which adenine could be involved directly. The discovery of hairpin ribozymes dependent on adenine for their reversible selfcleavage presents considerable biochemical and evolutionary interests because we show that RNA is able to use exogenous reactive molecules to enhance its own catalytic activity. Such a mechanism may have been a means by which the ribozymes of the RNA world enlarged their chemical repertoire.The RNA world theory assumes that modern life arose from molecular ancestors in which RNA molecules both stored genetic information and catalyzed chemical reactions (1-4). According to this scenario, ribozymes of the RNA world would have been able to self-replicate (5) and to control complex metabolisms with an expanded chemical repertoire (6, 7). Until recently, RNA catalysis was believed to be restricted to phosphate chemistry, but in vitro selection experiments and recent discoveries concerning natural ribozymes have demonstrated that the catalytic capacities of RNA are far more promising and exciting than previously anticipated (8 -13). However, in comparison with proteins, the chemical spectrum of ribozymes remains limited because of the limited chemical diversity of RNA, which is composed of only four different building blocks.Yet RNA could increase its range of functionalities by incorporating catalytic building blocks such as imidazole, thiol, and functional amino and carboxylate groups (14, 15). Moreover, primeval nucleotides were not necessarily restricted to standard nucleotides; modified nucleotides may have played a role in catalysis in the RNA world (16,17).Another way for RNA to increase its chemical diversity would consist in the binding of exogenous molecules carrying reactive groups and handling them as catalytic cofactors. We recently reported the isolation of new RNA aptamers able to bind adenine in a novel mode of purine recognition (18). Adenine is a likely prebiotic analog of histidine. Its catalytic capabilities are equivalent to histidine because of the presence of a free imidazole moiety (19 -21). It was previously shown that when adenine is placed in a favorable microenvironment, its catalytic efficiency is strongly enhanced (22-24). Such favorable microenvironments could result from adenine binding to RNA and thereby providing catalytic sites. In this perspe...

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Trapping malate dehydrogenase from the extremely halophilic archaeon Haloarcula marismortui in "dry" salt crystals protects the enzyme against thermal denaturation. Similar protection was not observed for the homologous mesophilic enzyme. In the case of transfer RNA molecules, high salt concentration plays a protective role against thermal degradation allowing activity to be recovered. The results are discussed in the context of exploring the fate of cell-free biological macromolecules in the environment and that of orienting the search for traces of life in planetary exploration.

The recent discovery of numerous catalytically active RNAs in various living species as well as the in vitro selection of a large series of RNA aptamers able to bind specifically various molecules such as metabolites and co-factors, emphasize the adaptability of RNAs through the plasticity of their secondary structure. Furthermore, all these observations give support to the "RNA world" hypothesis as a step in the primitive development of life on Earth. On this background, we used high pressure to study the mechanism of action of a model hairpin ribozyme which exhibits self-cleavage and ligation. The activation volume (DeltaV( not equal)) of the cleavage reaction (34+/-4 ml/mol) indicates that an important compaction of the RNA molecule occurs during the reaction and must be accompanied by a significant movement of water molecules . Indeed, such a release of 78+/-4 water molecules per RNA molecule could be measured by complementary osmotic shock experiments. These results are consistent with the information provided by the structural studies which indicate that two loops of the RNA molecule should come into contact for the reaction to occur . The high pressure study of a modified form of the ribozyme whose activity is strictly dependent on the presence of adenine as a co-factor should bring some information about the structural significance of this important DeltaV( not equal) of activation.