5. Some introns code for proteins that sponsor mobility

23.4 Ribozymes have various catalytic activities

The catalytic activity of group I introns was discovered by virtue of their ability to autosplice, but they are able to undertake other catalytic reactions in vitro. All of these reactions are based on transesterifications. We analyze these reactions in terms of their relationship to the splicing reaction itself.

Figure 23.6 Excision of the group I intron in Tetrahymena rRNA occurs by successive reactions between the occupants of the guanosine-binding site and substrate-binding site. The left exon is red, and the right exon is purple.

Figure 23.2 Self-splicing occurs by transesterification reactions in which bonds are exchanged directly. The bonds that have been generated at each stage are indicated by the shaded boxes.

The catalytic activity of a group I intron is conferred by its ability to generate a particular secondary and tertiary structure that creates active sites, equivalent to the active sites of a conventional (proteinaceous) enzyme. Figure 23.6 illustrates the splicing reaction in terms of these sites (this is the same series of reactions shown previously in Figure 23.2).

The P1 helix represents the formation of a substrate-binding site, in which the 3′ end of the first intron base pairs with the IGS in an intermolecular reaction. A guanosine-binding site is formed by sequences in P7. This site may be occupied either by a free guanosine nucleotide or by the G residue in position 414. In the first transfer reaction, it is used by free guanosine nucleotide; but it is subsequently occupied by G⁴¹⁴. The second transfer releases the joined exons. The third transfer creates the circular intron.

Figure 23.7 The position of the IGS in the tertiary structure changes when P1 is formed by substrate binding.

Binding to the substrate involves a change of conformation; before substrate binding, the 5′ end of the IGS is close to P2 and P8, but after binding, when it forms the P1 helix, it is close to conserved bases that lie between P4 and P5. The reaction is visualized by contacts that are detected in the secondary structure in Figure 23.7. In the tertiary structure, the two sites alternatively contacted by P1 are 37 Å apart, which implies a substantial movement in the position of P1.

Figure 23.3 The excised intron can form circles by using either of two internal sites for reaction with the 5 F end, and can reopen the circles by reaction with water or oligonucleotides.

The L-19 RNA is generated by opening the circular intron (shown as the last stage of the intramolecular rearrangements shown in Figure 23.3). It still retains enzymatic abilities. These resemble the activities involved in the original splicing reaction, and we may consider ribozyme function in terms of the ability to bind an intramolecular sequence complementary to the IGS in the substrate-binding site, while binding either the terminal G⁴¹⁴ or a free G-nucleotide in the G-binding site.

Figure 23.8 The L-19 linear RNA can bind C in the substrate-binding site; the reactive G-OH 3 F end is located in the G-binding site, and catalyzes transfer reactions that convert 2 C5 oligonucleotides into a C4 and a C6 oligonucleotide.

Figure 23.8 illustrates the mechanism by which it extends the oligonucleotide C₅ to generate a C₆ chain. The C₅ oligonucleotide binds in the substrate-binding site, while G⁴¹⁴ occupies the G-binding site. By transesterification reactions, a C is transferred from C₅ to the 3′-terminal G, and then back to a new C₅ molecule. Further transfer reactions lead to the accumulation of longer cytosine oligonucleotides. The reaction is a true catalysis, because the L-19 RNA remains unchanged, and is available to catalyze multiple cycles. The ribozyme is behaving as a nucleotidyl transferase.

Figure 23.9 Catalytic reactions of the ribozyme involve transesterifications between a group in the substrate-binding site and a group in the G-binding site.

Some further enzymatic reactions are characterized in Figure 23.9. The ribozyme can function as a sequence-specific endoribonuclease by utilizing the ability of the IGS to bind complementary sequences. In this example, it binds an external substrate containing the sequence CUCU, instead of binding the analogous sequence that is usually contained at the end of the left exon. A guanine-containing nucleotide is present in the G-binding site, and attacks the CUCU sequence in precisely the same way that the exon is usually attacked in the first transfer reaction. This cleaves the target sequence into a 5′ molecule that resembles the left exon, and a 3′ molecule that bears a terminal G residue. By mutating the IGS element, it is possible to change the specificity of the ribozyme, so that it recognizes sequences complementary to the new sequence at the IGS region (for review see Cech, 1990).

Altering the IGS, so that the specificity of the substrate-binding site is changed, enabling other RNA targets to enter, can be used to generate a ligase activity. An RNA terminating in a 3′ VOH is bound in the substrate site, and an RNA terminating in a 5′ VG residue is bound in the G-binding site. An attack by the hydroxyl on the phosphate bond connects the two RNA molecules, with the loss of the G residue.

The phosphatase reaction is not directly related to the splicing transfer reactions. An oligonucleotide sequence that is complementary to the IGS and terminates in a 3′ Vphosphate can be attacked by the G⁴¹⁴. The phosphate is transferred to the G⁴¹⁴, and an oligonucleotide with a free 3′ VOH end is then released. The phosphate can then be transferred either to an oligonucleotide terminating in 3′ VOH (effectively reversing the reaction) or indeed to water (releasing inorganic phosphate and completing an authentic phosphatase reaction).

Figure 23.10 Reactions catalyzed by RNA have the same features as those catalyzed by proteins, although the rate is slower. The K_M gives the concentration of substrate required for half-maximum velocity; this is an inverse measure of the affinity of the enzyme for substrate. The turnover number gives the number of substrate molecules transformed in unit time by a single catalytic site.

The reactions catalyzed by RNA can be characterized in the same way as classical enzymatic reactions in terms of Michaelis-Menten kinetics. Figure 23.10 analyzes the reactions catalyzed by RNA. The K_M values for RNA-catalyzed reactions are low, and therefore imply that the RNA can bind its substrate with high specificity. The turnover numbers are low, which reflects a low catalytic rate. In effect, the RNA molecules behave in the same general manner as traditionally defined for enzymes, although they are relatively slow compared to protein catalysts (where a typical range of turnover numbers is 10³ V10⁶).

How does RNA provide a catalytic center? Its ability seems reasonable if we think of an active center as a surface that exposes a series of active groups in a fixed relationship. In a protein, the active groups are provided by the side chains of the amino acids, which have appreciable variety, including positive and negative ionic groups and hydrophobic groups. In an RNA, the available moieties are more restricted, consisting primarily of the exposed groups of bases. Short regions are held in a particular structure by the secondary/tertiary conformation of the molecule, providing a surface of active groups able to maintain an environment in which bonds can be broken and made in another molecule. It seems inevitable that the interaction between the RNA catalyst and the RNA substrate will rely on base pairing to create the environment. Divalent cations (typically Mg²⁺) play an important role in structure, typically being present at the active site where they coordinate the positions of the various groups. They play a direct role in the endonucleolytic activity of virusoid ribozymes (see later).

The evolutionary implications of these discoveries are intriguing. The split personality of the genetic apparatus, in which RNA is present in all components, but proteins undertake catalytic reactions, has always been puzzling. It seems unlikely that the very first replicating systems could have contained both nucleic acid and protein.

But suppose that the first systems contained only a self-replicating nucleic acid with primitive catalytic activities, just those needed to make and break phosphodiester bonds. If we suppose that the involvement of 2′ VOH bonds in current splicing reactions is derived from these primitive catalytic activities, we may argue that the original nucleic acid was RNA, since DNA lacks the 2′ VOH group and therefore could not undertake such reactions. Proteins could have been added for their ability to stabilize the RNA structure. Then the greater versatility of proteins could have allowed them to take over catalytic reactions, leading eventually to the complex and sophisticated apparatus of modern gene expression.

Reviews

Cech, T. R. (1990). Self-splicing of group I introns. Ann. Rev. Biochem 59, 543-568.