Ribozymes, cofactors

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]. 

HammerheadRtbozyme. A small RNA molecule that catalyzes cleavage of the phosphodiester backbone of RNA is known as the hammerhead ribozyme. This ribozyme occurs namrally in certain vimses where it facihtates a site-specific self-cleavage at the phosphate and generates a 2 3 -cychc phosphate and a 5 -hydroxyl terminus. The reaction requires a divalent metal ion, such as or, as a cofactor. Whereas the... 

Cech and co-workers obtained a ribozyme which had been shortened via splicing, the L-19 RNA, from the system described above in the presence of guanosine or guanosine nucleotide as a cofactor, an intron with 414 nucleotides is cut out of the precursor rRNA, the result of splicing being L-19 RNA, which contains 395 nucleotides Cech was able to show that L-19 RNA is able to shorten and lengthen other oligonucleotide chains. 

A rather new approach for detecting metal ions with very high sensitivity and selectivity utilizes DNAzymes. DNAzymes are a special class of enzymes formed from DNA nucleotides. Compared to proteins and ribozymes, they are more stable, structurally simpler, and therefore cheaper. As DNAzymes often require metal ion cofactors, they are interesting sensing platforms for these metal ions [149]. 

Metal ions can function in several different ways as cofactors in ri-bozyme-catalyzed reactions, as described above, and proposed mechanisms for the reactions catalyzed by several ribozymes have taken advantage of such functions. Significant aspects of these functions of metal ions might be subsumed by nucleobases if their pKa values could be adjusted appropriately. The full details of the mechanisms of action of metalloenzymes remain to be elucidated. 

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. 

Catalytic antibodies, predicted by Jencks in 1969 and first discovered in 1986, can now be raised against a wide variety of haptens covering nearly every reaction. Catalytic antibodies are regarded as the best enzyme mimics, with very good selectivity, but almost always their catalytic efficiency is by far insufficient. Some natural RNA molecules act as catalysts with intrinsic enzyme-like activity which permits them to catalyze chemical reactions in the complete absence of protein cofactors. In addition, ribozymes identified through in-vitro selection have extended the repertoire of RNA catalysis. This versatility has lent credence to the idea that RNA molecules may have been central to the early stages of life on Earth. 

It has already been pointed out that a great deal of intracellular biochemistry is based on cofactors, with these cofactors, in turn, often being derived from nucleotides. However, while this indirectly implies the proficiency of ancient RNA catalysts, it does not prove that such catalysts could have existed. Although there are, for example, protein dehydrogenases and esterases, there are no modem ribozymes with similar activities. Just as engineering a ribozyme self-replicase will be an experimental demonstration that life could have arose via RNA, so the production of artificial ribozymes will be a demonstration that a metabolically complex RNA world may once have existed. 

Naturally occurring and synthetic ribozymes often require metal ion cofactors 2+... 

Figure 10.39 Selection of artificial ribozymes with amino acidic cofactors from the biosynthetic modified on DNAzyme libraries L26 and L27 the selection/amplification process to the L-his dependent ribozymes 10.55 and 10.56.

However, not all cleavage reactions of nucleic acids promoted by metal ions occur through direct involvement of metal ions in cleavage chemistry. For example, metal ion cofactors stabihze the catalytically active conformations of several ribozymes, but do not participate directly in catalysis. ... 

The hepatitis delta virus (HDV) ribozyme is a member of the class of small ribozymes and functions as a self-cleaving RNA sequence critical to the replication of the virus RNA genome (1, 8, 40). HDV ribozymes are proposed to employ several catalytic strategies that include an important example of general acid/base catalysis that involves a specific cytosine residue in the active site. Indeed, a milestone in our understanding of RNA catalysis was the observation that HDV and other small ribozymes could function in the absence of divalent metal ion cofactors, provided that high (molar) concentrations of monovalent ions are present (41, 42). These high monovalent ion concentrations are believed to stabilize the active RNA conformation, which implies that the primary role of divalent metal ions is in structural stabilization (42). 

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