RNA enzymes

Several features of these RNA enzymes, or ribozymes, lead to the realization that their biological efficiency does not challenge that achieved by proteins. First, RNA enzymes often do not fulfill the criterion of catalysis in vivo because they act only once in intramolecular events such as self-splicing. Second, the catalytic rates achieved by RNA enzymes in vivo and in vitro are... 

At first glance, it would seem that RNA enzymes, composed of fairly inert nucle-obases connected by a sugar-phosphate backbone, are simply not equipped with... 

Since the discovery that RNA molecules can possess catalytic activities, ribozymes have become a fascinating field both for academic researchers and the pharmaceutical industry. In this review, we emphasize the latest progress made in structure determination of ribozymes as well as the generation of DNA and RNA enzymes with novel catalytic properties by combinatorial approaches. 

ID 1MME3. Ribozymes, or RNA enzymes, catalyze a variety of reactions, primarily in RNA metabolism and protein synthesis The complex three-dimensional structures of these RNAs reflect the complexity inherent in catalysis, as described for protein enzymes in Chapter 6. (c) A segment of mRNA known as an intron, from the ciliated protozoan Tetrahymena thermophila (derived from PDB ID 1GRZ). This intron (a ribozyme) catalyzes its own excision from between exons in an mRNA strand (discussed in Chapter 26). 

In this chapter we examine the synthesis of RNA on a DNA template and the postsynthetic processing and turnover of RNA molecules. In doing so we encounter many of the specialized functions of RNA, including catalytic functions. Interestingly, the substrates for RNA enzymes are often other RNA molecules. We also describe systems in which RNA is the template and DNA the product, rather than vice versa. The information pathways thus come full circle, revealing that template-dependent nucleic acid synthesis has standard rules... 

The study of posttranscriptional processing of RNA molecules led to one of the most exciting discoveries in modern biochemistry—the existence of RNA enzymes. The best-characterized ribozymes are the self-splicing group I introns, RNase P, and the hammerhead ribozyme (discussed below). Most of the activities of these ribozymes are based on two fundamental reactions transesterification (Fig. 26-13) and phosphodiester bond hydrolysis (cleavage). The substrate for ribozymes is often an RNA molecule, and it may even be part of the ribozyme itself. When its substrate is RNA, an RNA cat-... 

The study of protein synthesis offers another important reward a look at a world of RNA catalysts that may have existed before the dawn of life as we know it. Researchers have elucidated the structure of bacterial ribosomes, revealing the workings of cellular protein synthesis in beautiful molecular detail. And what did they find Proteins are synthesized by a gigantic RNA enzyme ... 

The sequences of the amino acids in the chains from which proteins are constructed are encoded in the nucleotide sequences of DNA (deoxyribonucleic acid). The coding sequence for a protein in the DNA is found in the structural gene for that protein. The RNA enzymes are also encoded by DNA genes. A fourth major theme of the book deals with the nature of the genetic code used in DNA and with the processes by which cells read and interpret the code. It also includes study of the methods by which thousands of genes have been mapped to specific positions in chromosomes, isolated, cloned, and sequenced. 

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None of the detailed mechanisms to be discussed considers the macro-molecular association that may be involved in the action of RNase on high molecular weight polyribonucleotides. Preiss reported from light scattering studies that very large RNA-enzyme aggregates may be formed (393). Their significance for the catalytic mechanism is unknown. 

This behavior can be explained by the following kinetic considerations. The reaction leading to new RNA templates is catalyzed by a complex of enzyme and template. The affinity between these partners is so high that at the concentrations used every RNA molecule binds to an enzyme. The number of catalytically active complexes then rises exponentially until the RNA concentration becomes equal to the concentration of enzyme. At this point the enzyme is saturated with RNA. From now on the number of catalytically active RNA-enzyme complexes remains constant and the synthesis enters the linear phase, that is, the rate of appearance of new RNA molecules becomes constant. The new RNA molecules, now in excess over the enzyme, bind not only as templates but also, less strongly, at the site of synthesis. This leads to inhibition of synthesis by... 

Beaudry, A.A. Joyce, G.F. (1992). Directed evolution of an RNA enzyme. Science 257,635-641. Biebricher, C.K. (1987). Replication and evolution of short-chained RNA species by Q 3 replicase. Cold Spring Harbor Symposia on Quantitative Biology, Vol. 52, pp. 299-306. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 

Flory, C.M., Pavco, P.A., Jarvis, T.C., Lesch, M.E., Wincott, F.E., Beigelman, L. et al. (1996) Nuclease-resistant ribozymes decrease stromelysin mRNA levels in rabbit synovium following exogenous delivery to the knee joint. Proc. Natl. Acad. Sci. USA, 93, 754-758. Forster, A.C. and Altman, S. (1990) External guide sequences for an RNA enzyme. Science, 249, 783-786. 

Haseloff, J. and Gerlach, W.L. (1988) Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature, 334, 585-591. 

Mechanistic studies of RNA enzymes (ribozymes) and ribonucleoprotein (RNP) complexes such as the ribosome and telomerase, often seek to characterize RNA structural features, either dynamic or static, and relate these properties to specific catalytic functions. Many experimental techniques that probe RNA structure-function relationships rely upon site-specific incorporation of chemically modified ribonucleotides into the RNA of interest, often in the form of chemical cross-linkers to probe for sites of protein-RNA interaction or small organic fluorophores to measure dynamic structural properties of RNAs. The ability to arbitrarily modify any RNA molecule has been greatly enabled by modern RNA synthesis techniques however, there remains a practical size... 

Let us assume for now that an exponentially growing protocell with an enclosed autocatalytic metabolism could form and eventually evolve RNA enzymes. RNA enzymes would have co-evolved with the original metabolic pathways. After the evolution of protein enzymes, further takeover and transformation of pathways would have occurred. Pohorille and New [138] observed since there is no relationship between the RNA catalytic power of a given RNA and the protein for which that RNA can code, there is no clear path from the RNA world to the protein world. Therefore, protein cladis-tics can only make conclusions about metabolism after protein enzymes have... 

Catalytic RNAs, or ribozymes, are RNAs, which catalytically cleave covalent bonds in a target RNA. The catalytic site is the result of the conformation adopted by the RNA-RNA complex in the presence of divalent cations. Shortly thereafter, Altman and colleagues discovered the active role of the RNA component of RNase P in the process of tRNA maturation. This was the first characterization of a true RNA enzyme that catalyzes the reaction of a free substrate, i.e., possesses catalytic activity in trans (Guerrier et al. 1983). A variety of ribozymes, catalyzing intramolecular splicing or cleavage reactions, have subsequently been found in lower eukaryotes, viruses, and some bacteria. 

In 1986, Walter Gilbert formulated it explicitly in these terms If there are two enzymic activities associated with RNA, there may be more. And if there are activities among these RNA enzymes, or rihozymes, that can catalyse the synthesis of a new RNA molecule from precursors and an RNA template, then there is no need for protein enzymes at the beginning of evolution. One can contemplate an RNA world, containing only RNA molecules that serve to catalyse the synthesis of themselves. ... 

According to the free radical theory, the production of highly reactive oxygen free radicals cause progressive, random damage to DNA, RNA, enzymes, and other proteins, as well as unsaturated fatty acids and phospholipids, which eventually leads to cell death (H4). 

The majority of biologically active substances which have an important function seem to be labeled in some way. The labeling is genetically includol in the structure of certain macromolecules (i.e. RNA, enzymes, hormones) or the molecules are labeled in relation to the requirement of various metabolic processes according to well established rules. 

Site-specifically modified RNAs have been used in many applications to examine RNA stmcture-function relationships, RNA-protein interactions, RNA-ligand interactions, and RNA-catalysis mechanisms. Some earlier studies demonstrated the use of synthetic oligonucleotides to probe the roles of specific functional groups and detailed mechanisms in ribozyme catalysis (55). The synthesis of nucleoside analogs allows for a full-range of chemical diversity (e.g., inductive effects, space-filling capacity, etc.) to be explored, such that quantitative stmcture activity relationships can be determined for RNA enzymes and other biologically important RNAs (56). 

Nucleic acid selection methods have also been exploited for the development of novel RNA enzymes or ribozymes (58). An m-vitro-selected RNA that contains the modified nucleotide 5-(4-pyridylmethyl)-uridine (Table 1) can catalyze carbon-carbon bond formation in a Diels-Alder cycloaddition, with an 800-fold rate acceleration compared with a random RNA (49). Modified RNAs that contain the same uridine modification have also been selected to mediate metal-metal bond formation in the synthesis of palladium nanoparticles (59). Modified RNAs are likely to have many other applications as novel ribozymes that catalyze important biological reactions or can be used to create novel materials. 

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