6.7: Ribozymes - RNA Enzymes

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Learning Goals (ChatGPT o1, 1/30/25)

• Foundations of Catalysis by RNA (Ribozymes):

  • Explain the concept that catalysis is not exclusive to proteins and that RNA molecules can fold into defined secondary and tertiary structures to form active catalytic centers.
  • Describe the basic principles of electron pushing in chemical reactions, including the roles of electron donors (sources) and electron acceptors (sinks), and how these principles apply to ribozyme catalysis.

• Classification and Structural Diversity of Ribozymes:

  • List and classify the different types of ribozymes, including small self-cleaving ribozymes (hammerhead, viroid, hairpin, and riboswitches) as well as larger self-splicing introns (Group I and Group II) and RNP complexes (e.g., RNase P, spliceosome, ribosome).
  • Compare the sizes, structures, and functional roles of small versus large ribozymes, including their dependence on metal ions and other cofactors for catalytic activity.

• Mechanisms of Ribozyme Catalysis:

  • Illustrate the general mechanism of RNA-catalyzed phosphodiester bond cleavage, including the roles of general acid and base catalysis, SN2 transesterification, and transition state stabilization.
  • Analyze specific reaction mechanisms for well-characterized ribozymes (e.g., hammerhead, hairpin) by mapping out electron flow (arrow-pushing) and identifying critical nucleotides (e.g., G8, G12) that facilitate catalysis.

• Functional Examples and Applications:

  • Examine the catalytic strategies of ribozymes such as self-cleaving reactions in viroids and hairpin ribozymes, noting how they process their own RNA or cleave external substrates (cis versus trans catalysis).
  • Discuss the roles of larger ribozymes (Group I and II introns, spliceosomal introns, RNase P, and the ribosome) in RNA processing and gene expression, and compare their mechanisms to those of small self-cleaving ribozymes.

• Evolutionary Implications:

  • Evaluate the hypothesis that ribozymes may have preceded protein enzymes in early evolution, considering their dual role as both genetic material and catalysts.
  • Discuss the significance of ribozyme catalysis in the RNA world hypothesis and how modern ribozymes (and engineered artificial ribozymes) inform our understanding of early biocatalysis.

• Advanced Functional Ribozymes:

  • Explore specialized ribozymes such as the methyltransferase ribozyme (MTR1), detailing how small molecule cofactors (e.g., O6-methylguanine) assist in methyl transfer reactions and expand the catalytic repertoire of RNA.
  • Discuss how ribozymes integrate cofactor binding, structural dynamics, and catalytic mechanisms to perform complex chemical transformations.

• Analytical and Structural Techniques:

  • Identify and discuss the experimental methods (such as X-ray crystallography, cryo-EM, and interactive structural modeling via tools like iCn3D) used to elucidate ribozyme structures and catalytic mechanisms.
  • Interpret interactive models and figures to understand the active site organization and the role of key residues in ribozyme function.

By achieving these learning goals, students will be well-equipped to understand the chemical and biological principles underpinning ribozyme catalysis, appreciate the structural and mechanistic diversity of RNA enzymes, and recognize their evolutionary significance and practical applications in modern biochemistry.

 

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 to form unique 3D structures, with active sites with appropriate functional groups or nonprotein "cofactors" (metal ions, vitamin derivatives) participating 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 are presenting the section before Chapter 8 for a few reasons. Most readers have encountered the structures of RNA and DNA 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 the essential 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 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 is used for 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 30-150 nucleotides, while large ones can be a few thousand nucleotides in length. This translates into approximate molecule weights (using this formula for single-stranded RNA: (# nucleotides x 320.5) + 159.0) into 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-spicing introns, bacterial ribonuclease P, and the RNA in spliceosomes and ribosomes. Many are not true enzymes since they catalyze their cleavage, although some can cleave presented RNA substrates. Large ones act as true catalysts.

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

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 the cleavage of an internal phosphodiester bond (cis catalysis) or in a presented substrate (trans catalysis) by 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 engage in 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 a 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 a S_N2 reaction and attacks the δ⁺ phosphorous in the phosphodiesterase bond, forming a pentavalent, trigonal pyramidal sp³d hybridized intermediate/transition state, which collapses, breaking the phosphodiesterase bond between nucleotide N₁ and N₂. This is an inline mechanism: the incoming nucleophilic O in C2' and the exiting one on the O of 5' CH₂OH of nucleotide 2 are axial, separated by 180⁰. 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. It has a structure similar to the head of a hammerhead shark. 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 ribosome 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 manson 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 manson 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 is a bit different in the iCn3D model above. PDB requires sequential numbering, whereas ribozyme sequences are numbered in discontinuous ways. Core residues that are conserved 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 viral capsid protein. Some enter cells along with viruses and are called virusoids or viroidlike satellite RNAs. Intrastrand pairing occurs, and they are synthesized as tandem repeats containing multiple adjacent copies of the viroid. These repeats are cut and ligated to form the individual mature viroid by internal ribozyme sequences. 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

The satellite RNA of plant viruses encodes 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 required for binding (self or other RNA molecules) and stem B, required for catalysis. Self-cleavage in the hairpin ribozyme occurs in stem A between an A and G bases (which are splayed apart) when the 2' OH on the A attacks the phosphorous 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 the transesterification cleavage of RNA.

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 pg 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):

A novel use of ribozymes was recently reported by Winkler et al. They discovered that the 5' end of the mRNA of the gene glmS (from 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 pathway inhibition. 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 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 in length. Before we discuss introns, we'll provide a quick background on RNA splicing. There are two major types of self-splicing introns, 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 required 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 when removed. 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 splicesome 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 the 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 RNA), and higher plants and are in ribosomal RNA (rRNA), mRNA, and tRNA. 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 the splicing reaction. They have one active site that catalyzes the initial cleavage of the phosphodiester bond and the final religation after cleavage.

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 without 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 the 3′-splice site, joining 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 session 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 cyanobacteria) 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 that 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, introns in pre-mRNA are removed by splicing and subsequent exon ligation, releasing the intron as a branched lariat molecule. This reaction is performed by the spliceosome, a large nuclear ribonucleoprotein (RNP) complex. Spliceosomes remove introns and splice the exons of most nuclear genes. They are composed of 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 (snRNA), 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 through the catalytic action of one essential RNA subunit that displays ribozyme activity. It's found in most organisms. As with many ribozymes, the 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 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 in cartoon form. The enzyme cleaves the 5' head end of the precursors of transfer RNA (tRNA) molecules. The enzyme is a heterodimer in bacteria with one RNA and 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 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 the holder of the genetic information, it would need RNA polymerase activity. Artificial ribozymes with class I RNA ligase activity have been made. 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 represents the active site region of the ribozyme. Three catalytic residues A29, C30, and C47 have been identified in the actual ribozyme. In iCn3D model is of a mutant, C47U, which has no 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 Polymerases (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 attached the terminal phosphate of the G(1)TP substrate to form a pentavalent intermediate. The Mg cation is stabilized by oxygens on P 29 and 30 of the ribozyme. The Mg ion also stabilizes the developing charge in the transition state and in the charge in the intermediate. Stabilization of analogous divalent cations in the protein polymerase occurs through Asp side changes in the protein.

Ribosome

Protein synthesis from mRNA templates occurs on a ribosome, a nanomachine composed of proteins and ribosomal RNAs (rRNA). The ribosome is composed of two very large structural units. The smaller unit (termed 30S and 40S in bacteria and eukaryotes, respectively) coordinates the correct base pairing of the triplet codon on the mRNA with another small adapter RNA, transfer or tRNA, that brings a covalently connected 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 terminal 3'OH of the terminal A by a 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 down the mRNA, so the dipeptide-tRNA is now at the P or 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 bound mRNA on the 30S subunit and tRNAs covalently attached to 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 is 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 to stabilize the oxyanion transition state at the electrophilic carbon attack site is precisely located water, which is positioned at the oxyanion hole 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 in that the 2'OH is in position 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 perfect positioning of the substrates. 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 the 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 has recently been published (Ben-Shem et al). It is significantly larger (40%) with a mass of around 3x10⁶ 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 and generally found in nature catalyze phosphoryl transfer reactions and, for the ribosome, peptide bond formation. In vitro evolution can be used to drive new enzymatic functionalities, which would have been required in a RNA-only world that preceded the use of proteins 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 in the case of protein, small molecule cofactors that bind to a potential ribozyme might facilitate greater catalytic efficiency and an expanded repertoire of reaction types. Indeed, we have seen above how small molecules can bind to riboswitches. Figure \(\PageIndex{26}\) shows the reaction and structure of a methyltransferase one ribozyme (MTR1) that acts as a methyltransferase. The small ligand, O⁶-methylguanine, binds to the ribozymes 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. Subsections of the strands are named J12 (colored green), J23 (colored blue), and J31 (colored 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 the binding will raise the pK_a of the cytosine.

The methyl transfer reaction occurs in step 3 by the nucleophilic attack of A63 N1 on the methyl group of O⁶-methylguanine and the 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 active ribozyme would also require an exchange of the substrate strand to place unmethylated adenine at position 63.

The proposed mechanism is fully consistent with the structure of the MTR1 riboswitch and the effect of the 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 examines the fascinating world of ribozymes—RNA molecules that not only store genetic information but also function as catalysts. It emphasizes that the capacity for catalysis is not unique to proteins; RNA can fold into complex secondary and tertiary structures that support catalytic activity. The chapter is organized around several key themes:

1. Introduction to Ribozymes and Catalytic Principles:
   The chapter opens by discussing the fundamental concept that any molecule capable of forming defined three-dimensional structures and containing functional groups—such as those needed for general acid/base, electrostatic, nucleophilic, or transition state stabilization catalysis—can act as a catalyst. Ribozymes illustrate this principle by employing RNA's limited chemical repertoire to drive electron flow from donors (sources) to acceptors (sinks) during bond making and breaking.

2. Classification and Diversity:
   Ribozymes are classified based on size and function. The chapter highlights that there are twelve classes of ribozymes, including small self-cleaving RNAs (e.g., hammerhead, viroid, and hairpin ribozymes, as well as riboswitches like glmS) and large catalytic RNAs such as Group I and Group II introns, ribonuclease P, and the catalytic RNAs of the spliceosome and ribosome. These examples demonstrate both cis (self-cleavage) and trans catalytic activities, illustrating the diversity of mechanisms by which RNA enzymes can operate.

3. Mechanisms of Catalysis:
   Detailed mechanistic insights are provided into how ribozymes achieve catalysis. The chapter explains the SN2 transesterification mechanism typical of self-cleaving ribozymes, where a general base activates a 2'-OH nucleophile that attacks an adjacent phosphodiester bond, leading to bond cleavage and formation of a pentavalent transition state. Examples such as the hammerhead ribozyme illustrate how conserved nucleotides (e.g., G8 and G12) and even subtle conformational arrangements contribute to efficient catalysis.

4. Functional and Structural Examples:
   Using interactive models and structural diagrams, the chapter explores specific ribozyme examples:

   • Hammerhead Ribozymes: Discussed in terms of their conserved secondary structure and active site architecture, which enable efficient self-cleavage and even trans-cleavage of RNA substrates.
   • Viroids and Hairpin Ribozymes: These small RNAs demonstrate the ability of ribozymes to function in viral and satellite RNA contexts.
   • Glucosamine-6-phosphate Ribozyme (glmS): A unique example where the ribozyme is embedded in an mRNA and uses its own catalytic activity, modulated by binding of a small molecule cofactor, to regulate gene expression.
   • Group I and II Introns and Spliceosomal RNPs: These larger ribozymes exemplify how RNA catalysis is integrated into essential processes such as RNA splicing and ribosome function, hinting at an evolutionary legacy from an RNA world.

5. Evolutionary and Functional Significance:
   The chapter considers the evolutionary implications of ribozymes, noting that RNA-based catalysis may have preceded protein enzymes. This dual functionality of RNA—as both genetic material and catalyst—supports the RNA world hypothesis and underscores the importance of ribozymes in early biochemistry.

6. Integration with Modern Techniques:
   Finally, the chapter highlights the methods used to study ribozymes, such as X-ray crystallography, cryo-electron microscopy, and interactive modeling tools like iCn3D. These techniques have revealed detailed insights into ribozyme structures, active sites, and catalytic mechanisms, bridging the gap between theoretical models and experimental observations.

Overall, this chapter provides a comprehensive overview of ribozyme catalysis, demonstrating how RNA can serve as an enzyme through diverse structural strategies and catalytic mechanisms. It sets the stage for deeper exploration of RNA’s roles in modern biology and the evolutionary transition from RNA to protein catalysts.