Ribozyme evolution

The final challenge in modeling such systems will be to encapsulate an evolving ribozyme system [74,86,87] within vesicles formed from amphiphilic mixtures that are optimized for stability and permeability. It seems likely that one such mixture will have a set of properties that permit it to encapsulate a catalytic polymerase system and template, with sufficient permeability to allow substrate access to the enzyme at reasonable rates. Replication and ribozyme evolution would then occur in immensely large numbers... 

Biondi E, Branciamore S, Maurel MC, Gallori E Montmorillonite protection of an UV-irradiated hairpin ribozyme evolution of the RNA world in a mineral environment. BMC Evol Biol 2007, 7(Suppl 2) S2. 

The authors obtained an RNA ligase ribozyme using the method of in vitro evolution . Here, macromolecules are allowed to go through a series of synthetic cycles, which are followed by a proliferation phase, mutation and selection. As in Darwinian evolution, the goal is to carry out laboratory selection of molecules with certain required properties. 

Fig. 6.10 Schematic representation of the principle of the evolution of a ribozyme in a test tube. Several mutants are selected in each cycle and proliferate in the next step. Slightly modified after Culotta (1992)...

An introduction to the method of in vitro evolution is given by Wilson and Stoszak (1999). The RNA lipase ribozyme, with about 140 nucleotides (but without the pyrimidine base cytosine), folded in a defined structure and was able to reach a reaction rate 105 times higher than in the uncatalysed reaction. This result certainly surprised those biogenesis researchers who were critical of the RNA world but we do not know whether the result changed their attitude to it ... 

Szostak et al. worked on the basis of a simple cellular system which can replicate itself autonomously and which is subject to Darwinian evolution. This simple protocell consists of an RNA replicase, which replicates in a self-replicating vesicle. If this system can take up small molecules from its environment (a type of feeding ), i.e., precursors which are required for membrane construction and RNA synthesis, the protocells will grow and divide. The result should be the formation of improved replicases. Improved chances of survival are only likely if a sequence, coded by RNA, leads to better growth or replication of membrane components, e.g., by means of a ribozyme which catalyses the synthesis of amphiphilic lipids (Figs. 10.8 and 10.9). We can expect further important advances in the near future from this combination ( RNA + lipid world ). 

Three pieces of evidence are cited in support of an RNA World. Firstly, some 17 RNA ribozyme catalysts have been discovered that produce a diverse array of organic molecules, including peptide bond formation. Second, the ability to form the peptide bond and build proteins may lead to a complex evolution favoured by the proximity of proto-proteins, producing enhanced reaction efficiency. Finally, RNA is the intermediate in the biosynthesis of DNA, suggesting that it must have preceded DNA in the evolutionary process. 

Many examples of catalytic nucleic acids obtained by in vitro selection demonstrate that reactions catalyzed by ribozymes are not restricted to phosphodiester chemistry. Some of these ribozymes have activities that are highly relevant for theories of the origin of life. Hager et al. have outlined five roles for RNA to be verified experimentally to show that this transition could have occurred during evolution [127]. Four of these RNA functionalities have already been proven Its ability to specifically complex amino acids [128-132], its ability to catalyze RNA aminoacylation [106, 123, 133], acyl-transfer reactions [76, 86], amide-bond formation [76,77], and peptidyl transfer [65,66]. The remaining reaction, amino acid activation has not been demonstrated so far. 

Synthesis of the peptide bond takes place in the next step. Ribosomal peptidyl-transferase catalyzes (without consumption of ATP or GTP) the transfer of the peptide chain from the tRNA at the P site to the NH2 group of the amino acid residue of the tRNA at the A site. The ribosome s peptidyltransferase activity is not located in one of the ribosomal proteins, but in the 28 S rRNA. Catalytically active RNAs of this type are known as ribo-zymes (cf. p. 246). It is thought that the few surviving ribozymes are remnants of the RNA world"—an early phase of evolution in which proteins were not as important as they are today. 

A self-replicating polymer would quickly use up available supplies of precursors provided by the relatively slow processes of prebiotic chemistry. Thus, from an early stage in evolution, metabolic pathways would be required to generate precursors efficiently, with the synthesis of precursors presumably catalyzed by ri-bozymes. The extant ribozymes found in nature have a limited repertoire of catalytic functions, and of the ribozymes that may once have existed, no trace is left. To explore the RNA world hypothesis more deeply, we need to know whether RNA has the potential to catalyze the many different reactions needed in a primitive system of metabolic pathways. 

The search for RNAs with new catalytic functions has been aided by the development of a method that rapidly searches pools of random polymers of RNA and extracts those with particular activities SELEX is nothing less than accelerated evolution in a test tube (Box 26-3). It has been used to generate RNA molecules that bind to amino acids, organic dyes, nucleotides, cyano-cobalamin, and other molecules. Researchers have isolated ribozymes that catalyze ester and amide bond formation, Sn2 reactions, metallation of (addition of metal ions to) porphyrins, and carbon-carbon bond formation. The evolution of enzymatic cofactors with nucleotide handles that facilitate their binding to ribozymes might have further expanded the repertoire of chemical processes available to primitive metabolic systems. 

The enzymatic activity that catalyzes peptide bond formation has historically been referred to as peptidyl transferase and was widely assumed to be intrinsic to one or more of the proteins in the large ribosomal subunit. We now know that this reaction is catalyzed by the 23S rRNA (Fig. 27-9), adding to the known catalytic repertoire of ribozymes. This discovery has interesting implications for the evolution of life (Box 27-3). 

The discovery in the 1980s that RNA molecules often have catalytic properties and may serve as true enzymes (ribozymes Chapter 12) stimulated new thinking about evolution. Although RNA catalysts are not as fast as the best enzymes they are able to catalyze a wide variety of different reactions. Could it be that in the early evolution of organisms RNA provided both the genetic material and catalysts The "RNA world" would have been independent of both DNA and protein.a b Later DNA could have been developed as a more stable coding molecule and proteins could have evolved as more efficient catalysts. Plausible reactions by which both cytosine and uracil could have arisen in drying ponds on early Earth have been demonstrated.6... 

Lorsch, J. R., and J. W. Szostak, In vitro evolution of new ribozymes with polynucleotide kinase activity. Nature 371 31-36, 1994. 

Aptamers are nucleic acids which exhibit a defined structure due to their nucleotide sequence and therefore, are able to specifically bind selected targets [1] (aptus [lat.] = fitting, sticking to). Aptamers and likewise, ribozymes [2] and deoxyribozymes [3] are selected in vitro by screening nucleic acid libraries. Here we describe in detail the selection of aptamers by a process called SELEX (Systematic Evolution of Ligands by Exponential enrichment) [4]. 

Lehman, N. Joyce, G.F. (1993). Evolution in vitro Analysis of a lineage of ribozymes. Current Biology... 

Recombination, 136 Recombination activating genes (RAG1, RAG2), 19-22 Recombination signal sequences (RSS), 11-13, 18-22 Retinoic acid, 100, 104, 107, 108, 111 receptors, 108 Ribozymes, 160,164 evolution, 176 hammerhead, 162 Rhizobium, 199, 205, 210,211 RNA,... 

Paul, N., Springsteen, G., and Joyce, G. F. (2006). Conversion of a ribozyme to a deoxyribozyme through in vitro evolution. Chem. Biol. 13, 329-338. 

By simulating evolution in vitro it has become possible to isolate artificial ribozymes from synthetic combinatorial RNA libraries [1, 2]. This approach has great potential for many reasons. First, this strategy enables generation of catalysts that accelerate a variety of chemical reactions, e.g. amide bond formation, N-glycosidic bond formation, or Michael reactions. This combinatorial approach is a powerful tool for catalysis research, because neither prior knowledge of structural prerequisites or reaction mechanisms nor laborious trial-and-error syntheses are necessary (also for non-enzymatic reactions, as discussed in Chapter 5.4). The iterative procedure of in-vitro selection enables handling of up to 1016 different compounds... 

The first hypothesis is that RNAs have used available amino acids to evolve from an RNA only world towards a nucleic acid-protein world. This hypothesis is in agreement with the role of RNA in the translation machinery, as for example the fact that the peptidyl transferase activity of the ribosome has been associated with the nucleic acid moiety and not the protein moiety [16,17]. The driving force that guided the evolution from the RNA world towards the emergence of the translation machinery might have been that amino acids played a role of ribozyme cofactors [6,7]. 

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