Ribozyme activity

The hypothesis that our biological world built on the DNA-RNA-protein central dogma was preceded by an RNA world in which RNA molecules carried both the genetic information and executed the gene functions (through ribozyme activity) is now widely accepted [130]. However, it is also well recognized that RNA due to its vulnerability to hydrolysis - especially as a result of catalysis by divalent metal ions - would not have been able to evolve in a harsh pre-biotic environment Also the formation of RNA under presumed pre-biotic conditions is extremely inefficient It is not so far-fetched to propose that a peptide nucleic acid-like molecule may have been able to function as a form of pre-biotic genetic material since it... 

The a-amino group of the new aminoacyl-tRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA occupying the P site (peptidyl or polypeptide site). At initiation, this site is occupied by aminoacyl-tRNA mef. This reaction is catalyzed by a peptidyltransferase, a component of the 285 RNA of the 605 ribosomal subunit. This is another example of ribozyme activity and indicates an important—and previously unsuspected—direct role for RNA in protein synthesis (Table 38-3). Because the amino acid on the aminoacyl-tRNA is already activated, no further energy source is required for this reaction. The reaction results in attachment of the growing peptide chain to the tRNA in the A site. 

Soon after this report, the group of M. Yaros, also working in Boulder, was able to demonstrate ribozyme activity with a much higher performance (Illangsekare, 1995). Using a random mixture of many billions of RNA sequences, they selected one species which was able to catalyse the aminoacyl synthesis. In other words, the selected ribozyme aminoacylated its 2 (3 ) end when offered phenylalanyl-AMP the addition of Mg2+ and Ca2+ was necessary. The catalysed reaction was about 105 times faster than in the absence of ribozyme. Thus the group was able to show that a fundamental reaction of contemporary protein biosynthesis can also be catalysed by a ribozyme (see Sect. 5.3.2). The assiduous search for further activities continues. 

The hammerhead motif has a conserved secondary structure consisting of a three-way helical junction. The helical elements may vary in base sequence among species but thirteen bases at the three-way helical junction are conserved and essential for ribozyme activity. X-ray structures to be discussed below define a domain organization based on the tertiary folding observed in... 

Figure 6.17 Talo-5 C-methyl substituent at the hammerhead ribozyme active site.

A ribozyme activity that led to RNA-modifications that are analogous to the 5 -5 pyrophosphate caps of eukaryotic RNA transcripts was selected by Huang and Yarns [84]. Actually the author s intention was to isolate ribozymes which catalyze the formation of a mixed anhydride between an amino acid carboxylate and a 5 -terminal phosphate of an RNA, an activity that is chemically analogous to the activation of amino acids by ATP catalyzed by aminoacyl tRNA synthetases. However, while the selected ribozymes did... 

In our recent study of reaction kinetics, we observed an unusual phenomenon when we analyzed the activity of a hammerhead ribozyme as a function of the concentration of Na ions on a background of a low concentration of either Mn or Mg ions [82]. At lower concentrations of Na ions, Na ions had an inhibitory effect on ribozyme activity, whereas at higher concentrations, Na ions had a rescue effect. We propose that these observations can be explained if we accept the existence of two kinds of metal-binding site that have different affinities. Our data also support the two-phase folding theory [77-80, Fig. 7], in which divalent metal ions in the ri-bozyme-substrate complex have lower and higher affinities, as proposed by Lilley and coworkers on the basis of their observations of ribozyme complexes in the ground state. 

Our data indicate that ribozyme activity in a low concentration of divalent metal ions decreases dramatically at lower concentrations of NaCl (Fig. 8). 

This inhibitory effect of NaCl can be explained on the basis of the data from Horton et al. [81] that is described above. The Na ions remove Mn ions from the lower affinity site(s), which is somehow involved in the ribozyme activity, from the ribozyme-substrate complex. When Hammann et al. used ions in their NMR analysis, they noticed that the apparent Ka for the lower affinity ions depended on the concentration of Na ions [80]. An increase in the background concentration of NaCl from 10 mmol/1 to 50 mmol/1 weakened the affinity of the Mg ions for the complex. Their observations also reconcile with the observed inhibition by Na ions in our study, with the competitive removal of Mg /Mn ions from the ribozyme-substrate complex. 

As the concentration of NaCl is increased after ribozyme activity has reached a minimum, the activity of the ribozyme is restored (Fig. 8). This rescue cannot be explained in terms of the number of bound Mn ions since the number does not increase at higher concentrations of NaCl, according to Horton et al., as described above. It is likely that, at higher concentrations, Na ions can work to fold the complex into the active structure and, as a result, more efficient Mn ions, even at limited concentrations, can work as a catalyst (see below for details). More efficient Mn ions can work as a catalyst even at a limited concentrations. As we would anticipate from the structural role and inefficient catalytic activity of Na ions, several groups have reported that ribozyme-mediated cleavage reactions can occur even in the absence of divalent metal ions provided that the concentration of monovalent ions, such as Na" ions, is extremely high [64-66]. 

Ribozyme activities are not confined to splicing reactions. Among the large number of RNases, the most sophisticated enzymes are probably those involved in processing because they must attack preRNAs at specific sites. In his studies on... 

This creates a dilemma of a different sort. How are we to demonstrate ribozyme activities that no longer exist Perhaps we must design our own ribozymes to demonstrate the full potential for RNA to act as an enzyme. This approach is being used by Jack Szostak and Gerald Joyce, who indepen-... 

Klein, D. J., and Ferre-D Amare, A. R. (2006). Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 313, 1752—1756. 

Fig. 5.2.7. Schematic representation of folding into the hairpin ribozyme active conformation.

McCah, M.J., Hendry, P., and Jennings, P.A. (1992). Minimal sequence requirement for ribozyme activity. Proc. Natl. Acad. Sci. USA 89,5710 5714. 

Evidence for Acid/Base Catalysis in the Hepatitis Delta Virus Ribozyme Active Site... 

Figure 4 Direct and indirect models of metal ion activation of ribozyme catalysis. Model of classes of metal sites that influence ribozyme activity. The scheme on top depicts the binding of a metal ion important for ribozyme folding that binds distant from the active site but promotes a structural transition that permits catalysis or the binding of catalytic metal ions. Such binding interactions may result directly in overall folding or may merely foster small stmctural changes near the active site that are critical to ribozyme chemistry. The scheme below depicts the direct activation of ribozyme activity by binding of metal ions that interact with the reactive phosphate and are involved in metal ion catalysis. Adapted from Reference 38.

Figure 5 Proposed acid/base catalytic interactions in the HDV ribozyme active site. The cleavage site for the HDV ribozyme is shown with the nucleotide 5 to the site of bond cleavage shown in red and the nucleotide 3 to that site shown in blue. Two proposed mechanisms for the function of the C76 cytisine nucleobase and a hydrated active site metal ion are shown in which C76 acts as either an acid (left) or base (right) as described in the text.

Figure 6 Proposed divalent metal ion coordination interactions involved in metal ion catalysis by the GI intron ribozyme active site. The coordination interactions determined by substrate PS and 2 amino modification and quantitative metal rescue are depicted on the left. The individual interacting functional groups are shown in red. The observed metal ions (green spheres) in the crystal structure of the GI ribozyme active site is shown on the right and the metal ligands identified by functional studies are shown as red spheres. Adapted from Reference 56.

Polypeptides would have played only a limited role early in the evolution of life because their structures are not suited to self-replication in the way that nucleic acid structures are. However, polypeptides could have been included in evolutionary processes indirectly. For example, if the properties of a particular polypeptide favored the survival and replication of a class of RNA molecules, then these RNA molecules could have evolved ribozyme activities that promoted the synthesis of that polypeptide. This method of producing polypeptides with specific amino acid sequences has several limitations. First, it seems likely that only relatively short specific polypeptides could have been produced in this manner. Second, it would have been difficult to accurately link the particular amino acids in the polypeptide in a reproducible manner. Finally, a different ribozyme would have been required for each polypeptide. A critical point in evolution was reached when an apparatus for polypeptide synthesis developed that allowed the sequence of bases in an RNA molecule to directly dictate the sequence of amino acids in a polypeptide. A code evolved that established a relation between a specific sequence of three bases in RNA and an amino acid. We now call this set of three-base combinations, each encoding an amino acid, the genetic code. A decoding, or translation, system exists today as the ribosome and associated factors that are responsible for essentially all polypeptide synthesis from RNA templates in modem organisms. The essence of this mode of polypeptide synthesis is illustrated in Figure 2.8. 

An RNA Diels-Alderase ribozyme recently developed that catalyses the formation of carbon-carbon bonds between a tethered anthracene diene and a biotinylated maleimide dienophile. The ribozyme active site has been further characterised by chemical substitution of the diene and dienophile. It was shown that the diene must be an anthracene, and substitution only at specific sites is permitted. The dienophile must be a maleimide with an unsubstituted double bond. The RNA-diene interaction was found to be governed preferentially by stacking interactions. A ribozyme has been selected that catalyses the synthesis of dipeptides using an aminoacyl-adenylate substrate. The ribozyme catalysed the formation of 30 different dipeptides, many with rates similar to that of the Met-Phe dipeptide used in the selection process. 

Figure 1 Schematic view of the coordination sites in the hammerhead ribozyme active site. Upper left The coordination pattern of Mg2 in the C-site coordinated to G10.1 N7 and k9 Ow. Upper right The coordination pattern of Mg2 in the B-site bridging A9 02p and Cl.LO of the scissile phosphate. Lower Coordination sites for Na in the hammerhead ribozyme active site found in the RT-Na and dRT-Na simulations. Red numbers next to the coordination sites are the scores used to calculate the coordination index (see text). M, involves direct binding to A9 02P and C.1 02P and indirect binding to G10.1 N 7 through a water molecule. M2 involves direct binding to CT7 02- and C.l 02p M3 involves direct binding to CI7 02 and is positioned toward the outside of the active site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this book.)...

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