2.2.12: Ribozymes - RNA Enzymes

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Small Self-Cleaving Ribozymes: Mechanisms and Diversity

• Describe the universal SN2 transesterification mechanism shared by small self-cleaving ribozymes — in which a general base activates the 2'-OH of an upstream nucleotide to attack the adjacent phosphodiester bond in an inline geometry (nucleophilic O and leaving O separated by 180°), generating a pentavalent trigonal bipyramidal transition state/intermediate that collapses to produce a 2',3'-cyclic phosphate and a 5'-hydroxyl — and identify the catalytic residues acting as general base (G12 in hammerhead, C in HDV, A38 in hairpin) and general acid, and the role of transition state stabilization as the predominant mechanism in the hairpin ribozyme, which lacks sufficient functional groups for extensive acid/base catalysis.
• Explain the glucosamine-6-phosphate (GlcN6P) riboswitch/ribozyme (glmS) as an example of metabolite-responsive feedback control at the RNA level — describing how the product of the GlmS enzyme (glucosamine-6-phosphate synthetase) binds to the 5' ribozyme domain of its own mRNA and acts as a cofactor that activates self-cleavage, thereby inhibiting further synthesis of the enzyme — and identify this as a fundamentally different regulatory strategy from allosteric protein inhibition.

Group I and Group II Introns, and the Spliceosome

• Compare the splicing mechanisms of Group I and Group II introns — noting that Group I introns require an exogenous guanosine nucleophile (whose 3'-OH attacks the 5' splice site in the first transesterification) while Group II introns use an internal branch-point adenosine 2'-OH as the first nucleophile to form a branched lariat intermediate — explain the mechanistic and structural homology between Group II introns and spliceosomal introns that argues for their common evolutionary origin, and describe how large ribonucleoprotein complexes (the spliceosome, composed of U1, U2, U4/U6, and U5 snRNPs plus >100 proteins totaling 1.8 MDa) assist splicing while the RNA components (not the proteins) provide the catalytic activity.
• Explain how Group II introns can act as mobile genetic elements through retrotransposition — in which the excised branched lariat intron, with the help of an associated maturase/reverse transcriptase protein, can reinsert into DNA by reversing the splicing reaction — and contrast this with mitochondrial/chloroplast Group II introns that have lost mobility and function as classical introns.

Large Ribozymes and the RNA World

• Describe the catalytic mechanism of the ribosome as a ribozyme — explaining that no ribosomal protein is close enough to the peptidyl transferase center (PTC) to participate in general acid/base catalysis, that the rRNA itself catalyzes peptide bond formation primarily through intramolecular catalysis (the 2'-OH of the P-site tRNA participates in a proton shuttle) and transition state stabilization (a precisely positioned water molecule in the oxyanion hole, H-bonded to U2584 of the 23S rRNA, stabilizes the tetrahedral oxyanion intermediate at the electrophilic carbonyl carbon of the growing peptide) — and connect this to the RNA world hypothesis.
• Explain how the methyltransferase ribozyme MTR1 — in which the small molecule O⁶-methylguanine binds as a cofactor to activate the ribozyme active site, C10 acts as a general acid by donating its proton to guanine N1 while A63 N1 acts as the nucleophile in an SN2 attack on the methyl group — illustrates how small-molecule cofactors can expand the catalytic repertoire of RNA beyond phosphoryl transfer reactions, supporting the plausibility of an RNA world in which RNA was both the information carrier and a diverse metabolic catalyst.
• Explain the RNA world hypothesis — arguing that RNA probably preceded both DNA and proteins as the primary biological information molecule and catalyst because RNA can both carry sequence information and fold into active catalysts, that DNA required a protein enzyme (ribonucleotide reductase) encoded by DNA to be synthesized, and that the existence of ribozymes in all three domains of life and in fundamental processes (splicing, translation, tRNA processing) provides strong evidence for this primordial role.

 

Ribozymes

Any molecule that displays any of the catalytic motifs seen in the earlier chapters (general acid/base catalysis, electrostatic catalysis, nucleophilic catalysis, intramolecular catalysis, and transition state stabilization) can be a catalyst. So far, we have only examined protein catalysts. These can fold into unique 3D structures, with active sites that contain appropriate functional groups or nonprotein "cofactors" (metal ions, vitamin derivatives) that participate in catalysis. There is nothing special about the ability of proteins to do this. RNA can also form secondary and tertiary structures, as seen in Chapter 8. RNA molecules that act as enzymes are called ribozymes.

We present this section before Chapter 8 for a few reasons. Most readers have encountered RNA and DNA structures before. You most likely know about three different types of RNA: ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). Likewise, you know from introductory biology classes that the central dogma of biology- DNA, the holder of the genetic code- is transcribed into RNA, which is translated into a protein sequence. Finally, most have studied (even at the high school level) that the DNA of many species has exons and introns (intervening sequences), the latter of which are spliced out of RNA transcripts to form mature RNA. In this section, we will discuss the catalytic properties of ribozymes, so the introductory background we just mentioned, although important, takes a "second" seat to the chemistry of catalysis, which is the main topic of Chapter 6.

There are 12 classes of ribozymes

• Small self-cleaving RNAs (9 classes)
• Group I introns
• Group II introns
• Ribonuclease P

The large ribonucleoprotein nanoparticles, the spliceosome and ribosome, are also functionally ribozymes.

The term ribozyme refers to RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonuclease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in Chemistry in 1989 for their work on ribozymes.

Ribozymes can be categorized based on size. Small ones, which usually don't require metal ions for activity, vary from 30 to 150 nucleotides, while large ones can be a few thousand nucleotides in length. This translates into approximate molecular weights (using this formula for single-stranded RNA: (# nucleotides x 320.5) + 159.0) of 9800 for a 30-mer and 640,000 for a 2000-mer, typical of small and very large proteins/protein complexes, respectively. into two groups depending upon their size – small and large. Large ribozymes, which require metal ions for activity, can vary in size from a few hundred to several thousand nucleotides. Examples of small ones include hammerhead, viroid, hairpin, and riboswitch ribozymes. Large ones include type I and II self-splicing introns, bacterial ribonuclease P, and the RNA in spliceosomes and ribosomes. Many are not true enzymes since they catalyze their own cleavage, although some can cleave presented RNA substrates. Large ones act as true catalysts.

Since RNA can carry genetic information and act as enzymes, it probably evolved before proteins, which require nucleic acids for synthesis. In addition, DNA required a special enzyme (ribonucleotide reductase) encoded by DNA to reduce the 2'OH to a 2'H. Scientists have made artificial ribozymes to catalyze many reactions that require protein enzymes. We will explore several classes of ribozymes.

Small self-cleaving RNAs

We'll consider four small self-cleaving RNAs: hammerhead, viroid, hairpin ribozymes, and the glucosamine-6-phosphate riboswitch (glmS). All catalyze cleavage of an internal phosphodiester bond (cis catalysis) or a presented substrate (trans catalysis) via a transesterification reaction. Internal cis-catalysis reactions cleave the ribozyme into two fragments, activating their catalytic activity. They don't act as true catalysts, since they undergo only one cleavage cycle. Trans catalysis, in which a substrate RNA binds to and is cleaved by the ribosome, would be considered true catalysis.

The cleavage reaction in an internal cis cleavage is an S_N2 trans-esterification reaction, as shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): SN2 trans-esterification cleavage of an internal phosphodiester bond in small RNA ribozymes

In the reaction, an adjacent general base (:B) abstracts a proton of the C'2 OH of nucleotide N₁. The resulting 2' O- acts as a nucleophile in an SN2 reaction and attacks the δ+ phosphorus in the phosphodiester bond, forming a pentavalent, trigonal pyramidal sp3d hybridized intermediate/transition state, which collapses, breaking the phosphodiester bond between nucleotide N1 and N2. This is an in-line mechanism: the incoming nucleophilic O at C2' and the exiting one on the O of 5' CH₂OH of nucleotide 2 are axial, separated by 180 ⁰. A general acid, BH, facilitates the departure of the exiting nucleophile by its protonation. Bound metal ions may facilitate the reaction and can be considered cofactors, but might be more involved in maintaining a catalytically active structure. Self-cleaving small RNAs are also found in humans and may be part of long noncoding RNAs.

a. Hammerhead RNA

The hammerhead ribozyme is a small RNA ribozyme with a conserved core with three helical stems. Its structure resembles a hammerhead shark's head. One predicted secondary structure of a hammerhead ribozyme is shown below in Figure \(\PageIndex{2}\)

Figure \(\PageIndex{2}\): Predicted secondary structure and sequence conservation of the HH9 ribozyme found conserved from lizard to human genomes.

A possible trigonal pyramidal intermediate/transition state in the Hammerhead ribozyme is shown in Figure \(\PageIndex{3}\).

Figure \(\PageIndex{3}\): Role of G8 and G12 in active site of full-length Schistosoma mansoni hammerhead ribozyme. After Martick and Scott, Cell. 126, 2006 DOI:https://doi.org/10.1016/j.cell.2006.06.036

A deprotonated G-12 in the ribozyme probably acts as a general base that activates the 2'-OH to form the incoming nucleophile that attacks the trans-substrate RNA. The 2′-OH of G-8 in the ribozyme appears to hydrogen bond to the 5′-O of the departing nucleophile in the substrate, where bond scission occurs. The ribozyme increases the rate by 1000-fold.

Figure \(\PageIndex{4}\) shows an interactive iCn3D model of the full-length Schistosoma mansoni catalytically active hammerhead ribozyme (3dz5). This is an example of a ribozyme that acts in trans, as the cleaved phosphodiester bond is a bound RNA single-stranded substrate.

Figure \(\PageIndex{4}\): Full-length Schistosoma mansoni catalytically active hammerhead ribozyme (3dz5). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?qNBw2MY1RZ7xKogH7

The numbering system differs slightly in the iCn3D model above. PDB requires sequential numbering, whereas ribozyme sequences are numbered in discontinuous ways. Conserved core residues are given common numbers. However, the different projecting double-stranded RNA regions vary among ribozymes and are numbered differently. The G8 and G12 in Figure \(\PageIndex{3}\) are numbered G20 and G36, respectively.

b. Viroids

Viroids are small, single-stranded circular RNAs that infect plant cells. They are not packaged with the viral capsid protein. Some enter cells along with viruses and are called virusoids or viroidlike satellite RNAs. Intrastrand pairing occurs, and the viroids are synthesized as tandem repeats containing multiple adjacent copies. Internal ribozyme sequences cut and ligate these repeats to form the mature viroid. One example is the hepatitis delta virus (HDV), a satellite of the hepatitis B virus. A possible mechanism of catalysis of the hepatitis delta virus ribozyme involving general acid/base catalysis is shown in Figure \(\PageIndex{5}\).

Figure \(\PageIndex{5}\): General acid/base catalysis by Hepatitis delta virus (HDV) ribozyme

c. Hairpin ribozyme

Satellite RNAs of plant viruses encode hairpin ribozymes. They are about 50 nucleotides long and can cleave themselves internally or, in a truncated form, can cleave other RNA strands in a transesterification reaction. The structure consists of two domains, stem A, which is required for binding (self or other RNA molecules), and stem B, which is required for catalysis. Self-cleavage in the hairpin ribozyme occurs in stem A between an A and G base (which are splayed apart) when the 2' OH on the A attacks the phosphorus in the phosphodiester bond connecting A and G to form a pentavalent intermediate.

Rupert et al. solved the crystal structure of a hairpin ribozyme with a non-cleavable substrate analog containing a 2'-O-CH₃ group on the ribose. This acts as a nucleophile in RNA transesterification cleavage.

A38 in Stem B appears to be able to interact with the products (the cleaved A now in the form of a cyclic phosphodiester with itself) and the departing G. With a transition state pentavalent analog of the sessile A-G bond in which the phosphodiester linking A and G in the substrate is replaced with a pentavalent vanadate bridge between A and G. This is Illustrated in Figure \(\PageIndex{6}\).

Figure \(\PageIndex{6}\): Transition state binding stabilization of hairpin ribozyme (adapted from Rupert et al, Science, 298, p. 1423, 2002)

However, A 38 does not appear to react with the sessile A-G groups in the normal substrate, indicating that the main mechanism used by this ribozyme is transition-state binding. Since RNA molecules have fewer groups available for acid/base and electrostatic catalysis (compared to protein enzymes), ribozymes, presumably the earliest type of biological catalyst, probably use transition-state binding as their predominant mode of catalytic activity.

Figure \(\PageIndex{7}\) shows an interactive iCn3D model of the hairpin ribozyme in the catalytically active conformation (1M5K).

Figure \(\PageIndex{7}\): Hairpin ribozyme (human) in the catalytically active conformation (1M5K). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...goBWNHUbLU7my8

The hairpin ribozyme is shown in cyan sticks, and the inhibitor substrate is shown in brown sticks. The inhibitor contains a 2'-O-methyl adenosine (A2M12), so it can not be cleaved and instead acts as an inhibitor. The A38 shown in the catalytic mechanism is labeled A57 in the iCn3D.

d. Glucosamine-6-phosphate riboswitch (glmS):

Winkler et al. recently reported a novel use of ribozymes. They discovered that the 5' end of the mRNA of the glmS gene (found in Gram-positive bacteria) is a ribozyme. The GlmS gene encodes glucosamine-6-phosphate synthetase (GlmS), which catalyzes the reaction of fructose-6-phosphate and glutamine to glucosamine-6-phosphate (GlcN6P) and glutamate. This is the first committed step in bacterial cell wall synthesis. Glucosamine-6-phosphate binds to the ribozyme (3' end of the mRNA) and acts as a cofactor, leading to self-cleavage of the ribozyme. What an amazing mechanism for inhibiting a pathway! At high GlcN6P concentrations, it binds to the ribozyme, inhibiting its own synthesis. G40 in the active site appears to act as a general base. Figure \(\PageIndex{8}\) shows an interactive iCn3D model of the GlmS ribozyme bound to its catalytic cofactor, glucosamine-6-phosphate (GlcN6P) (2NZ4).

Figure \(\PageIndex{8}\): GlmS ribozyme bound to its catalytic cofactor glucosamine 6-phosphate (GlcN6P) (2NZ4) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...J19zdfwEujH4c8

Riboswitches are discussed in greater detail in Chapter 28.1: Regulation of Gene Expression in Bacteria.

Group I and Group II Introns

Introns present in RNA molecules must be removed to form mature RNA. About 80% of introns in humans are less than 200 nucleotides long, but some can be 10,000 or longer. Before we discuss introns, we'll briefly review RNA splicing. Two major types of self-splicing introns exist: Groups I and II. Other introns are removed by a ribonucleoprotein called the spliceosome. Some call these Group III introns. A simple two-step mechanism for the self-splicing Group I and II introns is shown in Figure \(\PageIndex{9}\).

Figure \(\PageIndex{9}\): Self-splicing of Group I and II introns. (after N.K. Tanner /FEMS Microbiology Reviews 23 (1999)

Both require first a scission of the RNA strand, followed by ligation of the two exons to form the mature RNA. Note that Group I introns require an external guanosine nucleophile, and the removed intron forms a circular RNA. In contrast, in Group II introns, an internal A residue acts as the first nucleophile in the scission reaction, and the intron, on removal, forms a branched lariat structure.

A simple two-step mechanism for Group II introns, which are spliced out from pre-mRNA in the nucleus by a ribonucleoprotein complex called the spliceosome, is shown in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\): Two steps of canonical RNA processing, from pre-mRNA to spliced RNA and the branched lariat intron. https://commons.wikimedia.org/wiki/F...g_reaction.svg. Creative Commons Attribution-Share Alike 3.0 Unported

Note that the mechanism is extremely similar to the auto-removal of Group II introns, suggesting an evolutionary relationship between the two.

Figure \(\PageIndex{11}\) below shows a more detailed view of the catalytic cycle of the spliceosome. Five small ribonucleoproteins (U1, U2, U4/U6, and U5 snRNPs) assemble on the nuclear pre-mRNA and facilitate intron removal, but the main mechanism involves ribozyme activity.

Figure \(\PageIndex{11}\): Step-wise spliceosome assembly from its U-snRNP components. Nguyen et al. Current Opinion in Structural Biology 2016, 36:48–57. http://dx.doi.org/10.1016/j.sbi.2015.12.005. CC BY license (http://creativecommons.org/licenses/by/4.0/).

Group I introns

These are found in bacteria, lower eukaryotes (including mitochondrial and chloroplast RNAs), and higher plants, and are present in ribosomal RNAs (rRNAs), mRNAs, and tRNAs. They are also found in Gram-positive bacteriophages (viruses that attack bacteria). As shown in Figure \(\PageIndex{9}\), they require guanosine as a cofactor and have a single active site for both scission and ligation to produce the mature mRNA, tRNA, or rRNA. Mg² is not needed for catalysis per se, but to maintain the correct tertiary structure of the ribozyme with the correct secondary structure. In Group I introns, a guanosine cofactor initiates splicing. They have one active site that catalyzes the initial phosphodiester cleavage and subsequent religation.

The Group I catalytic core from Tetrahymena thermophila has two domains. A cleft is formed between them when they pack, which can bind the short helix containing the 5' splice site. and the guanosine cofactor. This "active" site is pre-formed, devoid of substrates, similar to the active sites of protein enzymes. Figure \(\PageIndex{12}\) shows the secondary structure and the reaction of the group I intron ribozyme from Tetrahymena.

Figure \(\PageIndex{12}\): Secondary structure and reaction of the natural, cis-splicing group I intron ribozyme from Tetrahymena. Müller. Molecules 2017, 22(1), 75; https://doi.org/10.3390/molecules22010075. Creative Commons Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

The intron runs between the two triangles, which show the 5' (filled triangle) and 3' (open triangle) splice sites. The orange P1 helices contain the 5′-splice site (G:U). The P10 helix (grey), P9.0 helix (green), and the P9.2 helix (blue) are structured to present the appropriate 3′-splice site. During splicing (from (A) to (B)), the P1 helix extension (orange) is opened to expose the 3′-hydroxyl group of the terminal uridine at the 5′-splice site. The P10 helix then facilitates a conformational change, in which the 3′-exon (upper dashed line) is positioned adjacent to the 5′-exon (lower dashed line), allowing the nucleophilic attack of the 3′-uridine at the 3′-splice site, joining the 5′-exon and 3′-exon.

Figure \(\PageIndex{13}\) shows an interactive iCn3D model from cryoEM of the full-length holo L-16 ScaI Tetrahymena ribozyme (7EZ2).

Figure \(\PageIndex{13}\): Holo L-16 ScaI Tetrahymena ribozyme (7EZ2) (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...PhK3BSbFLDnDt8

Three sets of coplanar bases are found in the active site. These include C262, A263, and G312 (top layer, brown), G264, C311, and the ωG - labeled G3 in the iCn3D model- (cyan layer) and A261, A265, and U310 (bottom layer red). The ωG - labeled G3 is the nucleophile.

The structure is nearly identical to the apo-form of the ribozyme, with just an internal guide RNA sequence undergoing a large change and the guanosine binding site undergoing a small shift on binding RNA substrates.

Group 2 Introns

Group II introns are found in mRNA of bacteria and some Archaea, in rRNA, tRNA, and mRNA of chloroplasts and mitochondria, and fungi, plants, and protists. No Class 2 introns appear to be found in eukaryotic genomes. Some of these introns are in gene-encoding proteins, but most are in bacterial noncoding sequences. In Group II introns, an adenosine cofactor initiates the splicing reaction, as shown in Figure \(\PageIndex{9}\).

What's especially interesting about group II introns is that they can reinsert into DNA.  Hence, they can be considered mobile genetic elements. A maturase/reverse transcriptase enzyme (562 amino acids) is associated with the intron, which helps stabilize the active site of the intron for the reversible scission and ligation of the intron. Reintegration of the excised branched lariat intron into DNA is called retrotransposition (copy/paste). Mitochondrial and chloroplast Group 2 introns have lost mobility and act as classic introns.

The structures of a group II intron from Thermosynechococcus vestitus (a cyanobacterium) before and after integration have been determined. A branch-site domain VI helix swings 90°, enabling DNA integration. The maturase/revere transcriptase protein assists excision of the intron through the interaction of domain VI of that intron, which positions the key adenosine for branched lariat formation during forward splicing, as shown in Figure \(\PageIndex{9}\). The changes in the structure of the group II intron retroelement before (6ME0) and after DNA integration (6MEC) are shown in Figure \(\PageIndex{14}\).

Figure \(\PageIndex{14}\): Changes in the structure of the group II intron retroelement before (6ME0) and after DNA integration (6MEC)

The target DNA before integration is shown in cyan spacefill and orange spacefill after integration. The protein is shown as a colored cartoon. The Group II intron is 867 nucleotides, and the sense target DNA is 47 nucleotides in length.

Spliceosomal Introns

A comparison of Figure \(\PageIndex{9}\) through Figure \(\PageIndex{11}\) shows the similarities between spliceosomal introns and group II self-splicing introns. Spliceosomal and group II self-splicing introns are structurally and mechanistically homologous, right down to the stereochemistry of the splicing reaction. In eukaryotes, splicing removes introns from pre-mRNA and ligates exons, releasing the intron as a branched lariat. The spliceosome, a large nuclear ribonucleoprotein (RNP) complex, performs this reaction. Spliceosomes remove introns and splice the exons of most nuclear genes. They contain 5 kinds of small nuclear RNA (snRNA) molecules and over 100 different protein molecules. The RNA — not the protein — catalyzes the splicing reactions. The molecular details of the reactions are similar to those of Group II introns, which has led to speculation that this splicing machinery evolved from them. Figure \(\PageIndex{15}\) shows two views of the cryo-EM structure of the human-activated spliceosome (the Bact complex)

C

Figure \(\PageIndex{15}\): Cryo-EM structure of the human-activated spliceosome (the Bact complex). Zhang et al. https://www.nature.com/articles/cr201814.pdf. Creative Commons Attribution 4.0 Unported License. http:// creativecommons.org/licenses/by/4.0/

There are 52 proteins, three small nuclear RNAs (snRNAs), and one pre-mRNA. The total molecular mass is 1.8M. U2, U5, and U6 snRNAs are colored marine, orange, and green, respectively. Pre-mRNA is colored red. Figure \(\PageIndex{16}\) shows just the structural changes in RNA and protein components that occur between the early Bact complex (left panel) and the mature Bact complex (right panel).

Figure \(\PageIndex{16}\): Structural changes in RNA and protein components that occur between the early Bact complex (left panel) and the mature Bact complex of the human spliceosome.

Ribonuclease P

This enzyme is a ribonucleoprotein that cleaves RNA via the catalytic activity of an essential RNA subunit that displays ribozyme activity. It's found in most organisms. As with many ribozymes, activity is increased 2-3-fold with bound proteins that stabilize the folded ribozyme and help bind the preferred substrate, pre-tRNA. Figure \(\PageIndex{17}\) shows the bacterial RNase P ribozyme in complex with tRNA (3q1r).

Figure \(\PageIndex{17}\): Bacterial RNase P ribozyme in complex with tRNA (3q1r).

The tRNA is shown as a magenta cartoon/surface, and the protein is shown as a cartoon. The enzyme cleaves the 5' head end of transfer RNA (tRNA) precursors. In bacteria, the enzyme is a heterodimer with one RNA and one protein subunit.

Figure \(\PageIndex{18}\) shows a possible transition state with key residues involved in binding two Mg²⁺ ions in the active site. These ions are essential for catalysis as they stabilize the pentavalent intermediate/transition state.

Figure \(\PageIndex{18}\): Transition state of RNase P with key RNA active site

Figure \(\PageIndex{19}\) shows an interactive iCn3D model of the active site with key residues labeled of the bacterial RNase P ribozyme in complex with tRNA (3q1r).

Figure \(\PageIndex{19}\): Bacterial RNase P holoenzyme in complex with tRNA (3q1r) (Copyright; author via source).
Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...6SjVMa8g6K47x5

RNA Polymerase Ribozyme

If primordial RNA acted as a metabolic enzyme catalyst and held genetic information, it would need RNA polymerase activity. Researchers have made artificial ribozymes with class I RNA ligase activity. Figure \(\PageIndex{20}\) shows an interactive iCn3D model of the active site region of the Class I ligase ribozyme-substrate preligation complex, C47U mutant, Mg²⁺ bound ( 3R1L).

Figure \(\PageIndex{20}\): Active site of the Class I ligase ribozyme-substrate preligation complex, C47U mutant, Mg²⁺ bound ( 3R1L). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...EEQV3Mzc8Jzdv6

The blue stick represents just the 3' terminal adenosine end of the target substrate (5'UCCAGUA3') to which a new nucleoside would be added. The brown region represents the ribozyme's active site. Three catalytic residues, A29, C30, and C47, have been identified in the actual ribozyme. The iCn3D model is of a mutant, C47U, that lacks catalytic activity. The green sphere represents Mg²⁺ ions.

Figure \(\PageIndex{21}\) shows the active site residues and how they might facilitate stabilization of the pentavalent intermediate/transition state and the similarity of the active site to a protein RNA polymerase.

Figure \(\PageIndex{21}\): Comparison of Transition State Models of Ribozyme RNA ligase and a protein RNA Polymerase (after Schechner et al)

A divalent Mg²⁺ in the active site of the ribozyme enhances the nucleophilicity of the 3-OH on the primer, which attacks the terminal phosphate of the G(1)TP substrate to form a pentavalent intermediate. The Mg cation is stabilized by oxygens on P29 and P30 of the ribozyme. The Mg ion also stabilizes the developing charge in the transition state and in the intermediate. Stabilization of analogous divalent cations in the protein polymerase occurs through changes in the protein's Asp side chains.

Ribosome

Protein synthesis from mRNA templates occurs on a ribosome, a nanomachine composed of proteins and ribosomal RNAs (rRNA). The ribosome has two very large structural units. The smaller unit (termed 30S in bacteria and 40S in eukaryotes) coordinates correct base pairing between the triplet codon on the mRNA and another small adapter RNA, transfer RNA (tRNA), which brings a covalently bound amino acid to the site. Peptide bond formation occurs when another tRNA-amino acid molecule binds to an adjacent codon on mRNA. The tRNA has a cloverleaf tertiary structure with some intrastrand H-bonded secondary structure. The last three nucleotides at the 3' end of the tRNA are CpCpA. The amino acid is esterified to the 3'OH of the terminal A by the protein enzyme aminoacyl-tRNA synthetase.

Covalent amide bond formation between the second amino acid and the first, forming a dipeptide, occurs at the peptidyl transferase center on the larger ribosomal subunit (50S and 60S in bacteria and eukaryotes, respectively). The ribosome ratchets along the mRNA, so the dipeptide-tRNA is now at the P (Peptide) site.  It awaits a new tRNA-amino acid at the A or Amino site. The figure below shows a schematic of the ribosome with mRNA bound to the 30S subunit and tRNAs covalently attached to an amino acid (or the growing peptide) at the A and P sites, respectively. Figure \(\PageIndex{22}\) shows a cartoon model of the prokaryotic ribosome with bound mRNA, tRNAs, and the P and A sites.

Figure \(\PageIndex{22}\): Cartoon model of the prokaryotic ribosome with bound mRNA, tRNAs, and the P and A sites.

We present another, more detailed model of the ribosome complex illustrating protein synthesis in Figure \(\PageIndex{23}\).

Figure \(\PageIndex{23}\): Ribosome mRNA translation. https://en.Wikipedia.org/wiki/File:R...slation_en.svg. public domain by its author, LadyofHats

A likely mechanism (derived from crystal structures with bound substrates and transition state analogs) for forming the amide bond between a growing peptide on the P-site tRNA and the amino acid on the A-site tRNA is shown below. Catalysis does not involve any ribosomal proteins (not shown) since none are close enough to the peptidyl transferase center to provide amino acids that could participate in general acid/base catalysis, for example. Hence, the rRNA must act as the enzyme (i.e., it is a ribozyme). Initially, it was thought that a proximal adenosine with a perturbed pKa could, at physiological pH, be protonated/deprotonated and hence act as a general acid/base in the reaction. However, none was found. The most likely mechanism for stabilizing the oxyanion transition state at the electrophilic carbon attack site is a precisely positioned water molecule at the oxyanion hole, stabilized by H-bonds to uracil 2584 on the rRNA. The cleavage mechanism involves the concerted proton shuffle shown below. In this mechanism, the substrate (Peptide-tRNA) assists its own cleavage, as the 2'OH is positioned to initiate the protein shuttle mechanism. (A similar mechanism might occur to facilitate hydrolysis of the fully elongated protein from the P-site tRNA.) Of course, all of this requires precise substrate positioning. Isn't that what enzymes do best? The main mechanisms for catalysis of peptide bond formation by the ribosome (as a ribozyme) are intramolecular catalysis and transition-state stabilization by an appropriately positioned water molecule. These processes are illustrated in Figure \(\PageIndex{24}\).

Figure \(\PageIndex{24}\): Mechanism Peptide Bond Formation by the Ribosome

The crystal structure of the eukaryotic ribosome is known. It is significantly larger (40%) and has a mass of around 3×10⁶ Daltons. The 40S subunit has one rRNA chain (18) and 33 associated proteins, while the larger 60S subunit has three rRNA chains (25S, 5.8S, and 5S) and 46 associated proteins. The larger size of the eukaryotic ribosome facilitates more interactions with cellular proteins and greater regulation of cellular events. Figure \(\PageIndex{25}\) shows the two copies of the 80S yeast ribosome (4v88) presented to humble readers and authors alike.Figure \(\PageIndex{25}\): The 80S yeast ribosome (4v88). Each subunit is given a different color.

Figure \(\PageIndex{25}\): Structure of the yeast 80S ribosome.

Ribozyme methyltransferase

The ribozymes described above catalyze phosphoryl-transfer reactions and, for the ribosome, peptide bond formation. In vitro evolution can drive the evolution of new enzymatic functions that would have been required in an RNA-only world before proteins were used as catalysts. RNA ribozymes have only four bases that can be employed in binding and catalytic steps, compared to 20 amino acids that can serve the same function in proteins. However, as with protein cofactors, small-molecule cofactors that bind to a potential ribozyme might enhance catalytic efficiency and expand the repertoire of reaction types. Indeed, as shown above, small molecules can bind to riboswitches. Figure \(\PageIndex{26}\) shows the reaction and structure of a methyltransferase ribozyme (MTR1) that acts as a methyltransferase. The small ligand, O⁶-methylguanine, binds to the ribozyme and acts as a cofactor in the methylation of adenine 63 in the RNA.

Figure \(\PageIndex{26}\): Deng, J., Wilson, T.J., Wang, J. et al. Structure and mechanism of a methyltransferase ribozyme. Nat Chem Biol 18, 556–564 (2022). https://doi.org/10.1038/s41589-022-00982-z. Creative Commons Attribution 4.0 International License. Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0/

Panel a shows the chemical reaction in which the methyl group of the small ligand O⁶-methylguanine is transferred to N1 of Adenine 63 in the RNA.

Panel b shows the sequence of the MTR1 ribozyme as crystallized for the experiments. The RNA is a three-way junction composed of three arms, P1, P2, and P3. A GNRA tetraloop has been added to the end of the P3 helix so that the entire ribozyme comprises a single RNA strand. The strands' subsections are named J12 (green), J23 (blue), and J31 (red).

X-ray crystal structures of the ribozyme in the presence of the cofactor O⁶-methylguanine were determined. The final structure contained guanine and an A63-methylated adenosine (1MA), implying the methyl group of the O⁶-methylguanine had transferred to A63, leaving guanine bound in the active site. The structure of the guanine bound to the ribozyme is shown in Figure \(\PageIndex{27}\).

Figure \(\PageIndex{27}\): The four planes of nucleobase interactions in the core of the ribozyme. The four planes are composed of (from bottom to top) G12•C38, C11•G41, exogenous guanine hydrogen-bonded to C10, U45, and A63, and the triple interaction A9•A46•A40. Deng, J et al., ibid.

Figure \(\PageIndex{28}\) shows a hypothetical reaction mechanism.

Figure \(\PageIndex{28}\): A proposed catalytic mechanism for the ribozyme methyltransferase. Deng, J et al., ibid.

In step 1, the nucleobase of C10 becomes protonated, and in step 2, the O⁶-methylguanine becomes bound. However, these two steps are likely coordinated, as binding raises the pKa of cytosine.

In step 3, the methyl transfer occurs via nucleophilic attack by A63 N1 on the methyl group of O6-methylguanine, accompanied by coordinated breakage of the guanine O6–C bond. This involves a train of electron transfers, the movement of the proton from C10 N3 to guanine N1, and a concomitant shift of the positive charge from C10 to the N¹-methyladenine at position 63. In principle, the guanine can now be released as a product, although there is no evidence that this occurs with the present form of the ribozyme. Regeneration of an active ribozyme would also require an exchange of the substrate strand to place an unmethylated adenine at position 63.

The proposed mechanism is fully consistent with the structure of the MTR1 riboswitch and with the effects of substitutions at C10 and U45 on activity. The complete loss of methylation activity of the C10U variant is fully consistent with the proposed role as a general acid in addition to ligand binding.

Figure \(\PageIndex{29}\) shows an interactive iCn3D model of a methyltransferase ribozyme (7V9E).

Figure \(\PageIndex{19}\): A methyltransferase ribozyme (7V9E). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...LtyVP4DBnkEgH6

The parts of the ribozyme are named J12 (light green), J23 (cyan), and J31 (magenta). The active site residues, C10, U45, and 1MA63 (1-methyladenosine), are shown in CPK-colored sticks and labeled (disregard small separated spheres). The Ba²⁺ ion in the crystal structure is not displayed. The guanine is shown in spacefill.

Summary

This chapter extends the principles of enzyme catalysis — general acid/base catalysis, electrostatic (metal ion) catalysis, nucleophilic catalysis, intramolecular catalysis, and transition state stabilization — from protein enzymes to RNA-based catalysts called ribozymes, demonstrating that the chemical strategies for catalysis are not the exclusive property of proteins but arise from the three-dimensional folding of any polymer that can present appropriate functional groups and structural geometries.

Ribozymes are RNA molecules that function as enzymes. Tom Cech (Group I introns) and Sidney Altman (RNase P) discovered them in 1982, work recognized with the 1989 Nobel Prize in Chemistry. There are 12 recognized classes: nine classes of small self-cleaving RNAs (Group I and Group II introns and RNase P), plus the spliceosome and ribosome, as large ribonucleoprotein assemblies whose catalytic activity resides in their RNA components. Small ribozymes (~30–150 nt) typically do not require metal ions for catalysis and include the hammerhead, viroid, hairpin, and glmS ribozymes. Large ribozymes (hundreds to thousands of nucleotides) require metal ions, usually Mg²⁺, to maintain their tertiary structures and, in some cases, for direct catalytic participation, and include Group I and II introns, RNase P, and the ribosome.

Small self-cleaving ribozymes all catalyze the same fundamental reaction: an in-line S_N2 transesterification in which the 2'-OH of a nucleotide upstream of the scissile bond acts as the nucleophile, attacking the adjacent phosphorus in a geometry that places the incoming nucleophile and the leaving 5'-oxygen diametrically opposite (180°) through a pentavalent trigonal bipyramidal transition state/intermediate. The products are a 2',3'-cyclic phosphate and a 5'-hydroxyl. Catalytic strategies vary by ribozyme. In the hammerhead ribozyme (found conserved from lizard to human genomes), G12 acts as a general base to activate the 2'-OH, and the 2'-OH of G8 hydrogen bonds to the departing 5'-oxygen to facilitate leaving group departure, achieving a 1000-fold rate enhancement. In the hepatitis delta virus (HDV) ribozyme (a viroid of Hepatitis B virus), a cytosine residue acts as a general acid/base. In the hairpin ribozyme, A38 stabilizes the transition state through hydrogen bonding rather than acting as a general acid/base — consistent with the principle that RNA, having fewer diverse functional groups than the 20 amino acids of proteins, relies more heavily on transition state stabilization. The glmS riboswitch/ribozyme provides an elegant example of metabolite-responsive feedback control at the RNA level: the product glucosamine-6-phosphate binds to the 5' ribozyme region of the glmS mRNA and acts as a cofactor (with G40 as the general base) that activates self-cleavage, preventing further translation of the glucosamine-6-phosphate synthetase — a remarkable RNA-encoded feedback inhibition mechanism.

Group I and Group II introns are the major classes of self-splicing introns. Group I introns (found in bacteria, lower eukaryotes, plant organelles, and bacteriophages) require an exogenous guanosine cofactor whose 3'-OH initiates the first transesterification at the 5' splice site; the same active site then catalyzes the second transesterification (ligation of the two exons), releasing a linear intron that circularizes. Mg²⁺ is required for maintaining catalytically competent tertiary structure. Group II introns (found in bacteria, archaea, and organelles of fungi, plants, and protists) use an internal branch-point adenosine 2'-OH as the nucleophile in the first transesterification, forming a branched lariat intermediate — mechanistically identical to the reaction catalyzed by the spliceosome in eukaryotes. This deep mechanistic and structural homology strongly suggests that the spliceosome evolved from Group II introns, with proteins replacing some RNA functions over evolutionary time. Group II introns can additionally act as mobile genetic elements: the excised lariat, with an associated maturase/reverse transcriptase, can reinsert into new genomic sites by retrotransposition (a "copy-paste" mechanism). The spliceosome — a 1.8 MDa assembly of five snRNPs (U1, U2, U4/U6, U5) and over 100 proteins — removes introns from pre-mRNA in eukaryotic nuclei through the same two-step transesterification mechanism, with the U2, U5, and U6 snRNAs providing the catalytic activity while the proteins facilitate assembly, proofreading, and recycling.

RNase P is a ribonucleoprotein that processes the 5' ends of precursor tRNA molecules. It is found in most organisms; in bacteria it is a heterodimer of one RNA and one protein subunit, with the RNA providing catalytic activity. Its active site coordinates two Mg²⁺ ions that stabilize the pentavalent transition state in the phosphoryl transfer reaction, analogous to the two-metal-ion mechanism seen in many protein metalloenzymes.

The ribosome is the most biologically consequential ribozyme. Peptide bond formation occurs at the peptidyl transferase center (PTC) of the large ribosomal subunit (50S in bacteria, 60S in eukaryotes), where no ribosomal protein is positioned close enough to participate in general acid/base catalysis. The RNA alone catalyzes the reaction, primarily through two mechanisms: intramolecular catalysis, in which the 2'-OH of the P-site tRNA participates directly in a proton shuttle mechanism that facilitates both the nucleophilic attack of the A-site amino group on the P-site ester and departure of the tRNA leaving group; and transition state stabilization, in which a precisely positioned water molecule at the oxyanion hole (positioned by hydrogen bonds to U2584 of the 23S rRNA) stabilizes the tetrahedral oxyanion intermediate at the electrophilic carbonyl carbon.

The MTR1 methyltransferase ribozyme expands the known catalytic repertoire of RNA beyond phosphoryl transfer. O⁶-methylguanine binds as a small-molecule cofactor; C10 acts as a general acid by donating its proton to guanine N1; and A63 N1 acts as a nucleophile attacking the electrophilic methyl group in an SN2 reaction — demonstrating that RNA, like proteins, can use small-molecule cofactors to access reaction chemistries otherwise unavailable to the four RNA bases.

Collectively, the existence of ribozymes catalyzing phosphoryl transfer, peptide bond formation, splicing, RNA processing, and methyltransfer across all domains of life provides compelling evidence for the RNA world hypothesis: that early life used RNA as both the information storage molecule (now the role of DNA) and the primary catalyst (now largely the role of proteins), with DNA arising later (requiring the protein enzyme ribonucleotide reductase for its synthesis from ribonucleotide precursors) and proteins gradually supplanting RNA catalysts due to the greater chemical diversity of 20 amino acids over 4 bases.

(Summary written by Claude, Sonnet 4.6, Anthropic)