Ribozyme transesterification

In addition to the catalytic action served by the snRNAs in the formation of mRNA, several other enzymatic functions have been attributed to RNA. Ribozymes are RNA molecules with catalytic activity. These generally involve transesterification reactions, and most are concerned with RNA metabofism (spfic-ing and endoribonuclease). Recently, a ribosomal RNA component was noted to hydrolyze an aminoacyl ester and thus to play a central role in peptide bond function (peptidyl transferases see Chapter 38). These observations, made in organelles from plants, yeast, viruses, and higher eukaryotic cells, show that RNA can act as an enzyme. This has revolutionized thinking about enzyme action and the origin of life itself. 

Scheme 14-1. General in-line monoanionic mechanism of phosphodiester cleavage transesterification catalyzed by hairpin ribozyme the first proton transfer (PT1), the nucleophilic attack (Nu), and the exocyclic cleavage (Cl) steps are shown, and the Oip and O2p pathways are indicated by blue and red colored hydrogens, respectively. For the uncatalyzed model reaction in solution, the Ojp and O2p pathways are energetically equivalent...

Figure 14-7. Snapshots of the active site structures near the transition state of (top) the nucleophilic attack and (bottom) the exocyclic cleavage for the in-line monoanionic O2p mechanism of cleavage transesterification in the hairpin ribozyme. The yellow and red colored cartoon is for the substrate and ribozyme strands, respectively, and water molecules interacting with non-bridging oxygens and O5/ are shown...

The hepatitis delta virus (HDV) ribozyme is part of the circular single stranded RNA genome of the hepatitis delta virus which consists of a total of 1700 nucleotides. The HDV ribozyme is required for the processing of multimers of the genomic linear RNA transcripts to unit length by catalyzing a transesterification reaction that results in self cleavage [23]. 

The hammerhead ribozyme was the first identified motif to catalyze sequence-specific self-cleavage in plant virus satellite RNA molecules (Fig. 1). This motif facilitates magnesium ion-dependent transesterification with a turnover rate of about 1 s ... 

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 enzymatic activity of the L-19 IVS ribozyme results from a cycle of transesterification reactions mechanistically similar to self-splicing. Each ribozyme molecule can process about 100 substrate molecules per hour and is not altered in the reaction therefore the intron acts as a catalyst. It follows Michaelis-Menten kinetics, is specific for RNA oligonucleotide substrates, and can be competitively inhibited. The kcat/Km (specificity constant) is 10s m- 1 s lower than that of many enzymes, but the ribozyme accelerates hydrolysis by a factor of 1010 relative to the uncatalyzed reaction. It makes use of substrate orientation, covalent catalysis, and metalion catalysis—strategies used by protein enzymes. 

Self-splicing KNA. The precursor to the 26S rRNA of Tetrahymena contains a 413-nucleotide intron, which was shown by Cedi and coworkers to be selfsplicing, i.e., not to require a protein catalyst for maturation.581 582 This pre-rRNA is a ribozyme with true catalytic properties (Chapter 12). It folds into a complex three-dimensional structure which provides a binding site for free guanosine whose 3-OH attacks the phosphorus at the 5 end of the intron as shown in Fig. 28-18A, step a. The reaction is a simple displacement on phosphorus, a transesterification similar to that in the first step of pancreatic ribonuclease action (Eq. 12-25). The resulting free 3-OH then attacks the phosphorus atom at the other end of the intron (step b) to accomplish the splicing and to release the intron as a linear polynucleotide. The excised intron undergoes... 

Grosshans, C. A., and Cech, T. R. (1991). A hammerhead ribozyme allows synthesis of a new form of the Tetrahymena ribozyme homogeneous in length with a 3 end blocked for transesterification. Nucleic Acids Res. 19, 3875—3880. 

The so-called ribozymes (Box 22) were discovered in 1982 by T. Cech and S. Altman. The naturally occurring species catalyze predominantly one reaction type -hydrolysis or transesterification of phosphodiester bonds in RNA. A very important natural ribozyme is the ribosome. On the basis of X-ray crystallographic investigations it was recently shown that the active site for the peptide bond-formation reaction is composed exclusively of RNA. 

Ribozymes are RNA molecules that catalyze biochemical reactions (54). Ribozymes cleave single-stranded regions in RNA through transesterification or hydrolysis reactions that result in cleavage of phosphodiester bends (55). To date, several RNA catalytic mo-tife, group I introns, RNase P, and both ham-... 

The self-splicing introns and the RNA component of RNase P (which cleaves the 5 end of tRNA precursors) are two examples of ribozymes. These biological catalysts have the properties of true enzymes. They generally promote hydrolytic cleavage and transesterification, using RNA as substrate. Combinations of these reactions can be promoted by the excised group 1 intron of Tetrahymena rRNA, resulting in a type of RNA polymerization reaction. 

EXAMPLE 5.7 An allosterically (see Sec. 5.12) controlled ribozyme is the GlmS riboswitch (Fig. 5-3) in which a metabolite, guanine, adenine, or thiamine pyrophosphate, regulates the expression of a gene. The metabolite controls the activity of the ribozyme that cleaves the mRNA in which it resides. Such riboswitches are typically located in the 5 untranslated regions of mRNA. These ribozymes are members of a class that work by transesterification in which the adjacent 2 -hydroxyl group on the ribose backbone of an RNA residue is activated to attack the adjacent 3 -phospho-diester bond. 

In biology, transesterification reactions are catalyzed by biocatalysts, most commonly by enzymes (protein) and ribozymes (catalytic RNA). Various enzymes belonging to the main enzyme classes EC 2-6 catalyze reactions in which transesterification steps can virtually be fovmd, even though the overall reaction is something else. Herein, the focus is on enzymes that catalyze reactions leading to transesterification products as final products. Such enzymes typically belong to hydrolytic enzymes, also called hydrolases (EC 3). Few other biocatalysts, such as DNA topoisomerases (EC 5.99.1.x) and ribozymes, also catalyze transesterifications, but they are not considered. 

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