Ribozymes, self-splicing

It was first discovered in 1981 by Thomas Cech and coworkers that a primary transcript for the 26 S rRNA from the protozoan Tetrarhymena could cut, splice, and assemble itself into the mature 26 S rRNA. Subsequently it was shown that a specific rRNA could catalyze the assembly of RNAs other than itself and that this could occur in the absence of any protein. Such enzyme-like RNAs were termed ribozymes. Self-splicing has been found to occur for RNAs from a variety of species. Two main types of self-splicing mechanisms are known ... 

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... 

The discovery of self-splicing introns showed that RNA could catalyse chemical reactions. Yet, unlike proteins, RNA has no functional groups with pKa values and chemical properties similar to those considered to be important in protein-based enzymes. Steitz and Steitz (1993) postulated that two metal ions were essential for catalysis by ribozymes using a mechanism similar to DNA cleavage, in which a free 3 OH is produced. They proposed,... 

As stated previously in the introductory section, T. R. Cech and co-workers reported on the first catalytic RNA or ribozyme, the self-splicing intron of the... 

Enzymatically active ribonucleic acid segments (some of which are known as ribozymes) with the capacity to catalyze RNA self-splicing or peptide bond formation. The overall catalytic rate enhancement is around 10 ... 

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. 

The RNA world hypothesis requires a nucleotide polymer to reproduce itself. Can a ribozyme bring about its own synthesis in a template-directed manner The self-splicing rRNA intron of Tetrahymena (Fig. 26-26) catalyzes the reversible attack of a guanosine residue on the 5 splice junction (Fig. 26-37). If the 5 splice site and the internal guide sequence are removed from the intron, the rest of the intron can bind RNA strands paired with short oligonucleotides. Part of the remaining intact intron effectively acts as a template for the... 

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... 

Ribozymes, they act mostly in self-splicing of introns when nascent mRNA is altered to make mature mRNA... 

Lehnert, V., Jaeger, L., Michele, F., and Westhof, E. (1996). New loop-loop tertiary interactions in self-splicing introns of subgroup IC and ID A complete 3D model of the Tetrahymena thermophila ribozyme. Chem. Biol. 3, 993—1009. 

Karbstein, K., Lee, J., and Herschlag, D. (2007). Probing the role of a secondary structure element at the 5 - and splice sites in group I intron self-splicing The Tetrahymena L-16ScaI ribozyme reveals a new role for the G U pair in self-splicing. Biochemistry 46, 4861-4875. 

In Tetrahymena, the pre-rRNA molecule contains an intron that is removed by self-splicing (in the presence of guanosine, GMP, GDP or GTP) without the need for involvement of any protein. This was the first ribozyme discovered but many have since been reported. 

In at least one eukaryote, Tetmhymem, the pre-rRNA molecule contains an intron. Removal of the intron during processing of the pre-rRNA does not require the assistance of any protein Instead, in the presence of guanosine, GMP, GDP or GTP, the intron excises itself, a phenomenon known as selfsplicing. This was the first demonstration of ribozymes, that is, catalytic RNA molecules that catalyze specific reactions. The list of ribozymes is growing. For example, self-splicing introns have been discovered in some eukaryotic mRNAs and even peptidyl transferase, a key enzyme activity in protein synthesis, is now known to be a ribozyme (see Topic H2). 

Finally, novel nucleic acid catalysts have also been selected from random sequence pools (reviewed in Ref. 19). Joyce and co-workers have manipulated the function of the Group I self-splicing ribozyme, selecting variants that can utilize calcium or cleave DNA from partially randomized pools [20,21], Lorsch and Szostak [22] selected a polynucleotide kinase ribozyme from a completely random sequence pool that flanked a previously selected ATP binding site. Many of the novel ribozymes can catalyze reactions that are relevant to protein biosynthesis, bolstering arguments that translation may have arisen in a putative RNA world. For example, Lohse and Szostak [23] have selected ribozymes that can carry out an acyl transfer reaction, while Illangasekare et al. [24] have isolated a... 

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