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6.7: Ribozymes - RNA Enzymes

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    Any molecule that displays any of the catalytic motifs seen in the earlier chapters (general acid/base catalysis, electrostatic catalysis, nucleophilic catalysis, intramolecular catalysis, transition state stabilization) can be a catalyst. So far we have examined only protein catalysts. These can fold to form unique 3D structures, which can have active sites with 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 also can form complicated secondary and tertiary structures as we will see 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. They most likely know about three different types of RNA, ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA). Likewise they 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 the have studied (even at the high school level) that DNA of many species has exons and introns (intervening sequences), the latter which are spliced out of RNA transcripts to form mature RNA. In this chapter we will discuss the catalytic properties of ribozymes, so this introductory background, although important, takes a "second" seat to the chemistry of catalysis, which is the main topc 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 as well.

    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 metals ions for activity, vary from 30-150 nucleotides while large one can be a few thousand nucleotide in length. This translate into approximate molecule weight, using the formula for single-stranded RNA of (# 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 required 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. Examples of large one include type I and II self-spicing introns, bacterial ribonuclease P, as well as the RNA in spliceosomes and ribosomes. Many also are not really true enzymes since they catalyze their own cleavage, although some can cleave presented RNA substrates. Large one act as true catalysts.

    Since RNA can carry genetic information and also act as an enzyme, it mostly likely evolved in advance of proteins, which presently require DNA to encode them, and DNA which required a special enzyme (ribonucleotide reductase) encoded by DNA to reduce the 2'OH to an single 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- hammerhead, viroid and hairpin ribozymes as well as the glucosamine-6-phosphate riboswitch (glmS). All catalyze cleavage of an internal phosphodiester bond (cis catalysis) or in a presented substrate (trans catalysis) by a transesterifcation reaction. Internal cis catalysis reactions cleave the ribozyme into two fragments so it is no longer active. In that sense, they don't act as true catalysts since they engage in only one cycle of cleavage. 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 SN2 trans-esterification reaction as shown in Figure \(\PageIndex{1}\) below.

    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 a nucleophile in a effect SN2 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 in that the incoming nucleophilic O in C2' and the exiting one on the O of 5' CH2OH of nucleotide 2 are axial to each other separated by 1800. A general acid, BH, facilitates the departure of the exiting nucleophile by its protonation. Bound metal ions may act 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 human and may be part of long noncoding RNAs.

    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 \(\PageIndex{2}\)

    Hammerhead_HH( (RF02275).pngHammerhead_HH( (RF02275)_legend.png
    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 \(\PageIndex{3}\) below.

    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:

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

    Schistosoma manson catalytically active hammerhead ribozyme (3dz5).png

    NIH_NCBI_iCn3D_Banner.svg 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:

    The numbering system is a bit different in the iCn3D model above. PDB requires sequential numbering whereas ribozymes sequences are numbered in discontinuous way. Core residues the are conserved are given common number but the different projecting double-stranded RNA regions, which vary among ribozymes, are numbered differently. The G8 and G12 in Figure \(\PageIndex{3}\) above 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 ribozymes sequences. One example is the hepatitis delta virus (HDV), a satellite of the epatitis B virus. A possible mechanism of catalysis of the hepatitis delta virus ribozyme involving general acid/base catalysis is shown in Figure \(\PageIndex{5}\) below.

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

    c. Hairpin ribozyme

    Hairpin ribozymes are encoded by the satellite RNA of plant viruses. They are about 50 nucleotides long, and can cleave itself 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 an 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 not longe acts as a nucleophile in the transesterifcation 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, and also 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 \(\PageIndex{6}\) below.

    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 make more use of transition state binding as their predominant mode of catalytic activity.

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

    hairpin ribozyme in the catalytically-active conformation (1M5K)V2.png
    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:

    The hairpin ribozyme is shown in cyan sticks and the inhibitor substrate in brown sticks. The inhibitor contains a 2'-O-methyl adenosine (A2M12) so it can not be cleave 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 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 \(\PageIndex{8}\) below shows an interactive iCn3D model of GlmS Ribozyme Bound to Its Catalytic Cofactor, glucosamine 6 phosphate (GlcN6P) (2NZ4).

    GlmS Ribozyme - glucosamine 6 phosphate complex (2NZ4).png
    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:

    Group I and Group II Introns

    Introns present in RNA molecule must be removed to form the mature RNA. In humans, about 80% are less than 200 nucleotides but some can be 10,000 nucleotides or longer. 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 a ribonucleoprotein called the spliceosome. Some calle these Group III introns. A simple two step mechanism for the self-splicing Group I and II introns is shown in Figure \(\PageIndex{9}\) below.

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

    Both required first an 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 III introns that are spliced out from pre-mRNA in the nucleus by a ribonucleoprotein complex called the splicesome is shown in Figure \(\PageIndex{10}\) below.


    Figure \(\PageIndex{10}\): Two steps of canonical RNA processing, from pre-mRNA to spliced RNA and the branched lariat intron. 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.

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

    CryoEM structures of two spliceosomal complexesFig1.svg
    Figure \(\PageIndex{11}\): Step-wise spliceosome assembly from its U-snRNP components. Nguyen et al. Current Opinion in Structural Biology 2016, 36:48–57. CC BY license (

    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 bacteria 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 prdouce the mature mRNA, tRNA or rRNA. Mg2 is required not for catalysis per se but to maintain the correct tertiary structure of the ribozyme with correct secondary structure. In Group I introns, the splicing reaction is initiated by a guanosine cofactor. They have one active sites 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 with the 5' splice site. and the guanosine cofactor. This "active" site is preformed without substrates similar to the active sites of protein enzymes. Figure \(\PageIndex{12}\) below shows secondary structure and the reaction of the group I intron ribozyme from Tetrahymena.

    Fig1 transsplicingGpIIntronRibozymes.svg
    Figure \(\PageIndex{12}\): Secondary structure and reaction of the natural, cis-splicing group I intron ribozyme from Tetrahymena. Müller. Molecules 2017, 22(1), 75; Creative Commons Attribution (CC-BY) license (

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

    Holo L-16 ScaI Tetrahymena ribozyme (7EZ2).png

    NIH_NCBI_iCn3D_Banner.svg 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:

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

    What's especially interesting about group II introns is that they can reinsert in DNA so that can be considered to be 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. Reintegrtation of the excised branched lariat intron into DNA is called retrotransposition (copy/paste). Mitochondrial and chloroplast Group 2 intron have lost their mobility and act as classic introns.

    The structure 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 interaction of domain VI of that intron that positions the key adenosine for branched lariat formation during forward splicing, as shown in Figure \(\PageIndex{9}\) above. The changes in structure of the group II intron retroelement before (6ME0) and after DNA integration (6MEC) are shown in Figure \(\PageIndex{14}\) below.

    Figure \(\PageIndex{14}\): Changes in 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 in organ 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 \(\PageIndex{9}\) thorugh Figure \(\PageIndex{11}\) show 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 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. It is the RNA — not the protein — that catalyzes the splicing reactions. The molecular details of the reactions are similar to those of Group II introns, and this has led to speculation that this splicing machinery evolved from them. Figure \(\PageIndex{15}\) below shows two views of the cryo-EM structure of the human activated spliceosome (the Bact complex)

    CStructure of the human activated spliceosome_Fig1.svg

    Figure \(\PageIndex{15}\): Cryo-EM structure of the human activated spliceosome (the Bact complex). Zhang et al. Creative Commons Attribution 4.0 Unported License. http://

    There are 52 proteins, 3 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 RNA and protein components that occur between the early Bact complex (left panel) and the mature Bact complex (right panel).

    Structure of the human activated spliceosome-Fig2.svg
    Figure \(\PageIndex{16}\): Structural changes 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 enzymes is a ribonucleoprotein that cleaves RNA through the catalytic action of one essential RNA subunit that displays ribozyme activity. It's in found in most organisms. As with many ribozymes, the activity is increased 2-3 fold with the bound proteis, which stabilized the folded ribozyme and helps bind the preferred substrate which is pre-tRNA. Figure \(\PageIndex{17}\) below shows bacterial RNase P ribozyme in complex with tRNA (3q1r).

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

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

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

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

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

    Bacterial RNase P holoenzyme in complex with tRNA (3q1r).png

    NIH_NCBI_iCn3D_Banner.svg 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:

    RNA Polymerase Ribozyme

    If primordial RNA acted as both an metabolic enzyme catalysts as well as the holder of the genetic information, it would need to have RNA polymerase activity. Artifical ribozymes with class I RNA ligase activity have been made. Figure \(\PageIndex{20}\) below 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:

    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 represent 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 represent Mg2+ ions.

    Figure \(\PageIndex{21}\) below show 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 arotein RNA Polymerases (after Schechner et al)

    A divalent Mg2+ in the active site of the ribozyme enhances the nuclophilicity of the 3-OH on the primer, which attached the 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.


    Protein synthesis from a mRNA template 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 intrastranded 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 to the first, forming a dipeptide, occurs at the peptidyl transferase center, located 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 the P or Peptide site, awaiting 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 site, 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 yet another more detailed model of the the ribosome complex illustrating protein synthesis in Figure \(\PageIndex{23}\) below.

    Ribosome_mRNA_translation_en (1).svg
    Figure \(\PageIndex{23}\): Ribosome mRNA translation. public domain by its author, LadyofHats

    A likely mechanism (derived from crystal structures with bound substrates and transition state analogs) for the formation of 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 of the 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 acts 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 and 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 illustrate in Figure \(\PageIndex{24}\) below.

    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 mass of around 3x106 Daltons. The 40S subunit has one rRNA chain (18) and 33 associated proteins, while the larger 60S subunit has 3 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 give a different color.

    6.7: Ribozymes - RNA Enzymes is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski.

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