Splicing transesterification

Fig. 1.3 The currently accepted chemical mechanism of protein splicing. 1 N-S(O) acyl shift, 2 transesterification, 3 cleavage by succinimide formation, 4 S(0)-N acyl shift.

FIGURE 26-13 Transesterification reaction. This is the first step in the splicing of group I introns. Here, the 3 OH of a guanosine molecule acts as nucleophile. 

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. 

RNA Splicing What is the minimum number of transesterification reactions needed to splice an intron from an mRNA transcript Explain. 

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... 

The splicing mechanism, which is illustrated for this intein, is shown in the accompanying equations.1 1 Step a is an N —> S or N —> O acyl shift. This is followed by transesterification (step b) which involves either thioesters (as illustrated) or oxygen esters. Formation of a succinmide intermediate (step c) releases the intein and the spliced protein. The latter must undergo an S—> N or O—> N acyl shift (step d), and the succinimide in the extein must be hydrolyzed to complete the process. 

The mechanism of self-splicing in this case is somewhat different from that observed in the spliceosome reaction (fig. 28.21). First the 3 hydroxyl group of the guanosine cofactor attacks the phosphodiester bond at the 5 splice site. This is followed by another transesterification reaction in which the 3 hydroxyl group of the upstream RNA attacks the phosphodiester bond at the 3 splice site, thereby completing the splicing reaction. The final reaction products include the spliced rRNA and the excised oligonucleotide. 

The 3 -OH terminus of exon 1 then attacks the phosphodiester bond between the intron and exon 2. Exons 1 and 2 become joined, and the intron is released in lariat form. Again, this reaction is a transesterilication. Splicing is thus accomplished by two transesterification reactions rather than by hydrolysis followed by ligation. The first reaction... 

Figure 28.36. Self-Splicing Mechanism. The catalytic mechanism of the selfsplicing intron from Tetrahymena includes a series of transesterification reactions. [After T. Cech. RNA as an enzyme. Copyright 1986 hy Scientific American, Inc. All rights reserved.]...

Figure 29.32 Spliceosome assembly and action. U1 binds the 5 splice site and U2 binds to the branch point. A preformed U4-US-U6 complex then joins the assembly to form the complete spliceosome. The U6 snRNA re-folds and binds the 5 splice site, displacing Ul, Extensive Interactions between U6 and U2 displace U4. Then, in the first transesterification step, the branch-site adenosine attacks the S splice site, making a lariat Intermediate. US holds the two exons in close proximity, and the second transesterification takes place, with the S splice-site hydroxyl group attacking the 3 splice site. These reactions result in the mature spliced mRNA and a lariat form of the intron bound by U2. US. and U6. After T. Villa, j. A. Pletss. and C, Guthrie. Cell 109(2002) H9-1S2.]...

One possibility is that the 3 end of the poly(U) donor strand cleaves the phosphodiester bond on the 5 side of the insertion site. The newly formed 3 terminus of the acceptor strand then cleaves the poly(U) strand on the 5 side of the nucleotide that initiated the attack. In other words, a uridine residue could be added by two transesterification reactions. This postulated mechanism is similar to the one in RNA splicing. 

Splicing Occurs at Short, Conserved Sequences in Pre-mRNAs via Two Transesterification Reactions... 

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