E2. The Biggest Ribozyme - The Ribosome - Biology

Protein synthesis from a mRNA template occurs on a ribosome, a nanomachine composed of proteins and ribosomal RNAs (rRNA). 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: Prokaryotic Ribosme - P and A sites

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.

Figure: 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. The Jmol structure of a bacterial 70S ribosome showing mRNA and tRNA interactions is shown below.

Jmol: Updated 70 S Ribosome from Thermus Thermophilus showing mRNA and tRNA interactions Jmol14 (Java) | JSMol (HTML5) Not done; Fix

: Ribosome in Action

[ For the sake of answering a question that is long unanswered ]

Can two hammerhead ribozymes simultaneously cleave each other?

Yes, in principle they can.

two hammerhead ribozymes simultaneously cleave each other when both are expressed, but have these ribozymes cleave a target transcript when expressed alone.

This may be realized only when ribosomes have higher affinity for each other than for the target. As you said in the comment, inserting few mismatches in the ribozyme-target interaction may work. You may need to notice that since the two ribozymes are complementary to each other but cleave the same target, the target sequences will form a set of two mutually complementary regions. If separated well enough, there may not be much of a problem. From your design strategy I guess that UTRs are the best place to keep the ribozyme target sequence. If you keep both the target sequences in the 3'UTR (lets say), then they may form a hairpin structure, which may interfere in the targeting. So you can insert mismatches that also destabilize the possible hairpin.

Has it ever been shown in the literature that a hammerhead ribozyme was able to cleave the flanking arms of another ribozyme?

Not yet. Self cleaving examples are there but not mutually cleaving ones.


The Varkud satellite (VS) ribozyme is a naturally occurring RNA molecule, found in the bread mold Neurospora, that catalyzes the self-cleavage and ligation reactions necessary for the life cycle of the VS RNA. It is the largest known member of the small self-cleaving ribozymes and the only one in this class for which the structure is not known at atomic resolution. Using single-molecule fluorescence resonance energy transfer (smFRET), we have studied how the molecule’s global dynamics influence its function as a catalyst 1 . However, to gain more detailed insights into the VS ribozyme’s three-dimensional structure, dynamics, and catalytic mechanism, a high-resolution X-ray crystal structure of the complete molecule is crucial. To this end, we are trying to crystallize the VS ribozyme using a novel method of purifying the ribozyme in its native state without subjecting it to denaturing conditions. By avoiding the harsh conditions of traditional purification by denaturing gel electrophoresis, which can cause the molecule to refold into erroneous structures, we hope to isolate a conformationally homogeneous population more conducive to crystallization and structural studies.

Any questions? Then contact Nils Walter at [email protected]
Phone | (734) 615-2060
Fax | (734) 615-5524
Follow us on Twitter: @NilsWalterLab

This site is a member of the RNA WebRing
[ Join Now | Ring Hub ]


The sequence of DNA that encodes the sequence of the amino acids in a protein is transcribed into a messenger RNA chain. Ribosomes bind to messenger RNAs and use their sequences for determining the correct sequence of amino acids to generate a given protein. Amino acids are selected and carried to the ribosome by transfer RNA (tRNA) molecules, which enter the ribosome and bind to the messenger RNA chain via an anti-codon stem loop. For each coding triplet (codon) in the messenger RNA, there is a transfer RNA that matches and carries the correct amino acid for incorporating into a growing polypeptide chain. Once the protein is produced, it can then fold to produce a functional three-dimensional structure.

A ribosome is made from complexes of RNAs and proteins and is therefore a ribonucleoprotein complex. Each ribosome is composed of small (30S) and large (50S) components called subunits which are bound to each other:

  1. (30S) has mainly a decoding function and is also bound to the mRNA
  2. (50S) has mainly a catalytic function and is also bound to the aminoacylated tRNAs.

The synthesis of proteins from their building blocks takes place in four phases: initiation, elongation, termination, and recycling. The start codon in all mRNA molecules has the sequence AUG. The stop codon is one of UAA, UAG, or UGA since there are no tRNA molecules that recognize these codons, the ribosome recognizes that translation is complete. [5] When a ribosome finishes reading an mRNA molecule, the two subunits separate and are usually broken up but can be re-used. Ribosomes are ribozymes, because the catalytic peptidyl transferase activity that links amino acids together is performed by the ribosomal RNA. Ribosomes are often associated with the intracellular membranes that make up the rough endoplasmic reticulum.

Ribosomes from bacteria, archaea and eukaryotes in the three-domain system resemble each other to a remarkable degree, evidence of a common origin. They differ in their size, sequence, structure, and the ratio of protein to RNA. The differences in structure allow some antibiotics to kill bacteria by inhibiting their ribosomes, while leaving human ribosomes unaffected. In all species, more than one ribosome may move along a single mRNA chain at one time (as a polysome), each "reading" a specific sequence and producing a corresponding protein molecule.

The mitochondrial ribosomes of eukaryotic cells functionally resemble many features of those in bacteria, reflecting the likely evolutionary origin of mitochondria. [6] [7]

Ribosomes were first observed in the mid-1950s by Romanian-American cell biologist George Emil Palade, using an electron microscope, as dense particles or granules. [8] The term "ribosome" was proposed by scientist Richard B. Roberts in the end of 1950s:

During the course of the symposium a semantic difficulty became apparent. To some of the participants, "microsomes" mean the ribonucleoprotein particles of the microsome fraction contaminated by other protein and lipid material to others, the microsomes consist of protein and lipid contaminated by particles. The phrase "microsomal particles" does not seem adequate, and "ribonucleoprotein particles of the microsome fraction" is much too awkward. During the meeting, the word "ribosome" was suggested, which has a very satisfactory name and a pleasant sound. The present confusion would be eliminated if "ribosome" were adopted to designate ribonucleoprotein particles in sizes ranging from 35 to 100S.

Albert Claude, Christian de Duve, and George Emil Palade were jointly awarded the Nobel Prize in Physiology or Medicine, in 1974, for the discovery of the ribosome. [10] The Nobel Prize in Chemistry 2009 was awarded to Venkatraman Ramakrishnan, Thomas A. Steitz and Ada E. Yonath for determining the detailed structure and mechanism of the ribosome. [11]

The ribosome is a complex cellular machine. It is largely made up of specialized RNA known as ribosomal RNA (rRNA) as well as dozens of distinct proteins (the exact number varies slightly between species). The ribosomal proteins and rRNAs are arranged into two distinct ribosomal pieces of different sizes, known generally as the large and small subunit of the ribosome. Ribosomes consist of two subunits that fit together (Figure 2) and work as one to translate the mRNA into a polypeptide chain during protein synthesis (Figure 1). Because they are formed from two subunits of non-equal size, they are slightly longer in the axis than in diameter.

Bacterial ribosomes Edit

Bacterial ribosomes are around 20 nm (200 Å) in diameter and are composed of 65% rRNA and 35% ribosomal proteins. [12] Eukaryotic ribosomes are between 25 and 30 nm (250–300 Å) in diameter with an rRNA-to-protein ratio that is close to 1. [13] Crystallographic work [14] has shown that there are no ribosomal proteins close to the reaction site for polypeptide synthesis. This suggests that the protein components of ribosomes do not directly participate in peptide bond formation catalysis, but rather that these proteins act as a scaffold that may enhance the ability of rRNA to synthesize protein (See: Ribozyme).

The ribosomal subunits of bacteria and eukaryotes are quite similar. [16]

The unit of measurement used to describe the ribosomal subunits and the rRNA fragments is the Svedberg unit, a measure of the rate of sedimentation in centrifugation rather than size. This accounts for why fragment names do not add up: for example, bacterial 70S ribosomes are made of 50S and 30S subunits.

Bacteria have 70S ribosomes, each consisting of a small (30S) and a large (50S) subunit. E. coli, for example, has a 16S RNA subunit (consisting of 1540 nucleotides) that is bound to 21 proteins. The large subunit is composed of a 5S RNA subunit (120 nucleotides), a 23S RNA subunit (2900 nucleotides) and 31 proteins. [16]

Ribosome of E. coli (a bacterium) [17] : 962
ribosome subunit rRNAs r-proteins
70S 50S 23S (2904 nt) 31
5S (120 nt)
30S 16S (1542 nt) 21

Affinity label for the tRNA binding sites on the E. coli ribosome allowed the identification of A and P site proteins most likely associated with the peptidyltransferase activity labelled proteins are L27, L14, L15, L16, L2 at least L27 is located at the donor site, as shown by E. Collatz and A.P. Czernilofsky. [18] [19] Additional research has demonstrated that the S1 and S21 proteins, in association with the 3′-end of 16S ribosomal RNA, are involved in the initiation of translation. [20]

Archaeal ribosomes Edit

Archaeal ribosomes share the same general dimensions of bacteria ones, being a 70S ribosome made up from a 50S large subunit, a 30S small subunit, and containing three rRNA chains. However, on the sequence level, they are much closer to eukaryotic ones than to bacterial ones. Every extra ribosomal protein archaea have compared to bacteria has an eukaryotic counterpart, while no such relation applies between archaea and bacteria. [21]

Eukaryotic ribosomes Edit

Eukaryotes have 80S ribosomes located in their cytosol, each consisting of a small (40S) and large (60S) subunit. Their 40S subunit has an 18S RNA (1900 nucleotides) and 33 proteins. [22] [23] The large subunit is composed of a 5S RNA (120 nucleotides), 28S RNA (4700 nucleotides), a 5.8S RNA (160 nucleotides) subunits and 46 proteins. [16] [22] [24]

eukaryotic cytosolic ribosomes (R. norvegicus) [17] : 65
ribosome subunit rRNAs r-proteins
80S 60S 28S (4718 nt) 49
5.8S (160 nt)
5S (120 nt)
40S 18S (1874 nt) 33

During 1977, Czernilofsky published research that used affinity labeling to identify tRNA-binding sites on rat liver ribosomes. Several proteins, including L32/33, L36, L21, L23, L28/29 and L13 were implicated as being at or near the peptidyl transferase center. [25]

Plastoribosomes and mitoribosomes Edit

In eukaryotes, ribosomes are present in mitochondria (sometimes called mitoribosomes) and in plastids such as chloroplasts (also called plastoribosomes). They also consist of large and small subunits bound together with proteins into one 70S particle. [16] These ribosomes are similar to those of bacteria and these organelles are thought to have originated as symbiotic bacteria [16] Of the two, chloroplastic ribosomes are closer to bacterial ones than mitochrondrial ones are. Many pieces of ribosomal RNA in the mitochrondria are shortened, and in the case of 5S rRNA, replaced by other structures in animals and fungi. [26] In particular, Leishmania tarentolae has a minimalized set of mitochondrial rRNA. [27] In contrast, plant mitoribosomes have both extended rRNA and additional proteins as compared to bacteria, in particular, many pentatricopetide repeat proteins. [28]

The cryptomonad and chlorarachniophyte algae may contain a nucleomorph that resembles a vestigial eukaryotic nucleus. [29] Eukaryotic 80S ribosomes may be present in the compartment containing the nucleomorph. [ citation needed ]

Making use of the differences Edit

The differences between the bacterial and eukaryotic ribosomes are exploited by pharmaceutical chemists to create antibiotics that can destroy a bacterial infection without harming the cells of the infected person. Due to the differences in their structures, the bacterial 70S ribosomes are vulnerable to these antibiotics while the eukaryotic 80S ribosomes are not. [30] Even though mitochondria possess ribosomes similar to the bacterial ones, mitochondria are not affected by these antibiotics because they are surrounded by a double membrane that does not easily admit these antibiotics into the organelle. [31] A noteworthy counterexample, however, includes the antineoplastic antibiotic chloramphenicol, which successfully inhibits bacterial 50S and eukaryotic mitochondrial 50S ribosomes. [32] The same of mitochondria cannot be said of chloroplasts, where antibiotic resistance in ribosomal proteins is a trait to be introduced as a marker in genetic engineering. [33]

Common properties Edit

The various ribosomes share a core structure, which is quite similar despite the large differences in size. Much of the RNA is highly organized into various tertiary structural motifs, for example pseudoknots that exhibit coaxial stacking. The extra RNA in the larger ribosomes is in several long continuous insertions, [34] such that they form loops out of the core structure without disrupting or changing it. [16] All of the catalytic activity of the ribosome is carried out by the RNA the proteins reside on the surface and seem to stabilize the structure. [16]

High-resolution structure Edit

The general molecular structure of the ribosome has been known since the early 1970s. In the early 2000s, the structure has been achieved at high resolutions, of the order of a few ångströms.

The first papers giving the structure of the ribosome at atomic resolution were published almost simultaneously in late 2000. The 50S (large prokaryotic) subunit was determined from the archaeon Haloarcula marismortui [35] and the bacterium Deinococcus radiodurans, [36] and the structure of the 30S subunit was determined from Thermus thermophilus. [15] These structural studies were awarded the Nobel Prize in Chemistry in 2009. In May 2001 these coordinates were used to reconstruct the entire T. thermophilus 70S particle at 5.5 Å resolution. [37]

Two papers were published in November 2005 with structures of the Escherichia coli 70S ribosome. The structures of a vacant ribosome were determined at 3.5 Å resolution using X-ray crystallography. [38] Then, two weeks later, a structure based on cryo-electron microscopy was published, [39] which depicts the ribosome at 11–15 Å resolution in the act of passing a newly synthesized protein strand into the protein-conducting channel.

The first atomic structures of the ribosome complexed with tRNA and mRNA molecules were solved by using X-ray crystallography by two groups independently, at 2.8 Å [40] and at 3.7 Å. [41] These structures allow one to see the details of interactions of the Thermus thermophilus ribosome with mRNA and with tRNAs bound at classical ribosomal sites. Interactions of the ribosome with long mRNAs containing Shine-Dalgarno sequences were visualized soon after that at 4.5–5.5 Å resolution. [42]

In 2011, the first complete atomic structure of the eukaryotic 80S ribosome from the yeast Saccharomyces cerevisiae was obtained by crystallography. [22] The model reveals the architecture of eukaryote-specific elements and their interaction with the universally conserved core. At the same time, the complete model of a eukaryotic 40S ribosomal structure in Tetrahymena thermophila was published and described the structure of the 40S subunit, as well as much about the 40S subunit's interaction with eIF1 during translation initiation. [23] Similarly, the eukaryotic 60S subunit structure was also determined from Tetrahymena thermophila in complex with eIF6. [24]

Ribosomes are minute particles consisting of RNA and associated proteins that function to synthesize proteins. Proteins are needed for many cellular functions such as repairing damage or directing chemical processes. Ribosomes can be found floating within the cytoplasm or attached to the endoplasmic reticulum. Their main function is to convert genetic code into an amino acid sequence and to build protein polymers from amino acid monomers.

Ribosomes act as catalysts in two extremely important biological processes called peptidyl transfer and peptidyl hydrolysis. [43] The "PT center is responsible for producing protein bonds during protein elongation". [43]

Translation Edit

Ribosomes are the workplaces of protein biosynthesis, the process of translating mRNA into protein. The mRNA comprises a series of codons which are decoded by the ribosome so as to make the protein. Using the mRNA as a template, the ribosome traverses each codon (3 nucleotides) of the mRNA, pairing it with the appropriate amino acid provided by an aminoacyl-tRNA. Aminoacyl-tRNA contains a complementary anticodon on one end and the appropriate amino acid on the other. For fast and accurate recognition of the appropriate tRNA, the ribosome utilizes large conformational changes (conformational proofreading). [44] The small ribosomal subunit, typically bound to an aminoacyl-tRNA containing the first amino acid methionine, binds to an AUG codon on the mRNA and recruits the large ribosomal subunit. The ribosome contains three RNA binding sites, designated A, P and E. The A-site binds an aminoacyl-tRNA or termination release factors [45] [46] the P-site binds a peptidyl-tRNA (a tRNA bound to the poly-peptide chain) and the E-site (exit) binds a free tRNA. Protein synthesis begins at a start codon AUG near the 5' end of the mRNA. mRNA binds to the P site of the ribosome first. The ribosome recognizes the start codon by using the Shine-Dalgarno sequence of the mRNA in prokaryotes and Kozak box in eukaryotes.

Although catalysis of the peptide bond involves the C2 hydroxyl of RNA's P-site adenosine in a proton shuttle mechanism, other steps in protein synthesis (such as translocation) are caused by changes in protein conformations. Since their catalytic core is made of RNA, ribosomes are classified as "ribozymes," [47] and it is thought that they might be remnants of the RNA world. [48]

In Figure 5, both ribosomal subunits ( small and large ) assemble at the start codon (towards the 5' end of the mRNA). The ribosome uses tRNA that matches the current codon (triplet) on the mRNA to append an amino acid to the polypeptide chain. This is done for each triplet on the mRNA, while the ribosome moves towards the 3' end of the mRNA. Usually in bacterial cells, several ribosomes are working parallel on a single mRNA, forming what is called a polyribosome or polysome.

Cotranslational folding Edit

The ribosome is known to actively participate in the protein folding. [49] [50] The structures obtained in this way are usually identical to the ones obtained during protein chemical refolding however, the pathways leading to the final product may be different. [51] [52] In some cases, the ribosome is crucial in obtaining the functional protein form. For example, one of the possible mechanisms of folding of the deeply knotted proteins relies on the ribosome pushing the chain through the attached loop. [53]

Addition of translation-independent amino acids Edit

Presence of a ribosome quality control protein Rqc2 is associated with mRNA-independent protein elongation. [54] [55] This elongation is a result of ribosomal addition (via tRNAs brought by Rqc2) of CAT tails: ribosomes extend the C-terminus of a stalled protein with random, translation-independent sequences of alanines and threonines. [56] [57]

Ribosomes are classified as being either "free" or "membrane-bound".

Free and membrane-bound ribosomes differ only in their spatial distribution they are identical in structure. Whether the ribosome exists in a free or membrane-bound state depends on the presence of an ER-targeting signal sequence on the protein being synthesized, so an individual ribosome might be membrane-bound when it is making one protein, but free in the cytosol when it makes another protein.

Ribosomes are sometimes referred to as organelles, but the use of the term organelle is often restricted to describing sub-cellular components that include a phospholipid membrane, which ribosomes, being entirely particulate, do not. For this reason, ribosomes may sometimes be described as "non-membranous organelles".

Free ribosomes Edit

Free ribosomes can move about anywhere in the cytosol, but are excluded from the cell nucleus and other organelles. Proteins that are formed from free ribosomes are released into the cytosol and used within the cell. Since the cytosol contains high concentrations of glutathione and is, therefore, a reducing environment, proteins containing disulfide bonds, which are formed from oxidized cysteine residues, cannot be produced within it.

Membrane-bound ribosomes Edit

When a ribosome begins to synthesize proteins that are needed in some organelles, the ribosome making this protein can become "membrane-bound". In eukaryotic cells this happens in a region of the endoplasmic reticulum (ER) called the "rough ER". The newly produced polypeptide chains are inserted directly into the ER by the ribosome undertaking vectorial synthesis and are then transported to their destinations, through the secretory pathway. Bound ribosomes usually produce proteins that are used within the plasma membrane or are expelled from the cell via exocytosis. [58]

In bacterial cells, ribosomes are synthesized in the cytoplasm through the transcription of multiple ribosome gene operons. In eukaryotes, the process takes place both in the cell cytoplasm and in the nucleolus, which is a region within the cell nucleus. The assembly process involves the coordinated function of over 200 proteins in the synthesis and processing of the four rRNAs, as well as assembly of those rRNAs with the ribosomal proteins.

The ribosome may have first originated in an RNA world, appearing as a self-replicating complex that only later evolved the ability to synthesize proteins when amino acids began to appear. [59] Studies suggest that ancient ribosomes constructed solely of rRNA could have developed the ability to synthesize peptide bonds. [60] [61] [62] In addition, evidence strongly points to ancient ribosomes as self-replicating complexes, where the rRNA in the ribosomes had informational, structural, and catalytic purposes because it could have coded for tRNAs and proteins needed for ribosomal self-replication. [63] Hypothetical cellular organisms with self-replicating RNA but without DNA are called ribocytes (or ribocells). [64] [65]

As amino acids gradually appeared in the RNA world under prebiotic conditions, [66] [67] their interactions with catalytic RNA would increase both the range and efficiency of function of catalytic RNA molecules. [59] Thus, the driving force for the evolution of the ribosome from an ancient self-replicating machine into its current form as a translational machine may have been the selective pressure to incorporate proteins into the ribosome's self-replicating mechanisms, so as to increase its capacity for self-replication. [63] [68] [69]

Ribosomes are compositionally heterogeneous between species and even within the same cell, as evidenced by the existence of cytoplasmic and mitochondria ribosomes within the same eukaryotic cells. Certain researchers have suggested that heterogeneity in the composition of ribosomal proteins in mammals is important for gene regulation, i.e., the specialized ribosome hypothesis. [70] [71] However, this hypothesis is controversial and the topic of ongoing research. [72] [73]

Heterogeneity in ribosome composition was first proposed to be involved in translational control of protein synthesis by Vince Mauro and Gerald Edelman. [74] They proposed the ribosome filter hypothesis to explain the regulatory functions of ribosomes. Evidence has suggested that specialized ribosomes specific to different cell populations may affect how genes are translated. [75] Some ribosomal proteins exchange from the assembled complex with cytosolic copies [76] suggesting that the structure of the in vivo ribosome can be modified without synthesizing an entire new ribosome.

Certain ribosomal proteins are absolutely critical for cellular life while others are not. In budding yeast, 14/78 ribosomal proteins are non-essential for growth, while in humans this depends on the cell of study. [77] Other forms of heterogeneity include post-translational modifications to ribosomal proteins such as acetylation, methylation, and phosphorylation. [78] Arabidopsis, [79] [80] [81] [82] Viral internal ribosome entry sites (IRESs) may mediate translations by compositionally distinct ribosomes. For example, 40S ribosomal units without eS25 in yeast and mammalian cells are unable to recruit the CrPV IGR IRES. [83]

Heterogeneity of ribosomal RNA modifications plays an important role in structural maintenance and/or function and most mRNA modifications are found in highly conserved regions. [84] [85] The most common rRNA modifications are pseudouridylation and 2’-O methylation of ribose. [86]

Chemically Engineered Ribosomes: A New Frontier in Synthetic Biology

Author(s): Anna Chirkova, Matthias Erlacher, Ronald Micura, Norbert Polacek Innsbruck Biocenter, Medical University of Innsbruck, Division of Genomics and RNomics, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria., Austria


Journal Name: Current Organic Chemistry

Volume 14 , Issue 2 , 2010


Chemically modified RNA nucleotides have been introduced in the past into various ribozymes in order to understand RNA folding and the mechanism of RNA catalysis. Recently the ribosome, the largest natural ribozyme known to date, has been added to the list of enzymes amenable to synthetic biology. The chemically engineered ribosomes were active in various functional assays including single-turnover peptidyl transfer reaction as well as in vitro translation assays. Solid-phase synthesis of several non-natural nucleotide analogs and their subsequent introduction into the catalytic center of the ribosome, revealed the ribose 2-OH at position A2451 of 23S ribosomal RNA as key functional group for amide bond synthesis. By altering the chemical characteristics of the ribose at A2451 by replacing its 2-OH with selected functional groups demonstrated that hydrogen donor capability is essential for efficient transpeptidation. These findings in combination with data that accumulated over the past years allowed to propose a comprehensive model for peptide bond synthesis in which the A2451 2-OH directly assists in positioning one of the tRNA substrates via hydrogen-bond formation and thus supports amide bond synthesis via a proton shuttle mechanism. It is conceivable that cell-free translation systems employing rationally designed chemically engineered ribosomes can be established in the near future to produce peptides and proteins harboring unnatural amino acids.

Current Organic Chemistry

Title: Chemically Engineered Ribosomes: A New Frontier in Synthetic Biology


Author(s):Anna Chirkova, Matthias Erlacher, Ronald Micura and Norbert Polacek

Affiliation:Innsbruck Biocenter, Medical University of Innsbruck, Division of Genomics and RNomics, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria.

Abstract: Chemically modified RNA nucleotides have been introduced in the past into various ribozymes in order to understand RNA folding and the mechanism of RNA catalysis. Recently the ribosome, the largest natural ribozyme known to date, has been added to the list of enzymes amenable to synthetic biology. The chemically engineered ribosomes were active in various functional assays including single-turnover peptidyl transfer reaction as well as in vitro translation assays. Solid-phase synthesis of several non-natural nucleotide analogs and their subsequent introduction into the catalytic center of the ribosome, revealed the ribose 2-OH at position A2451 of 23S ribosomal RNA as key functional group for amide bond synthesis. By altering the chemical characteristics of the ribose at A2451 by replacing its 2-OH with selected functional groups demonstrated that hydrogen donor capability is essential for efficient transpeptidation. These findings in combination with data that accumulated over the past years allowed to propose a comprehensive model for peptide bond synthesis in which the A2451 2-OH directly assists in positioning one of the tRNA substrates via hydrogen-bond formation and thus supports amide bond synthesis via a proton shuttle mechanism. It is conceivable that cell-free translation systems employing rationally designed chemically engineered ribosomes can be established in the near future to produce peptides and proteins harboring unnatural amino acids.

The Ribosome Challenge to the RNA World

An RNA World that predated the modern world of polypeptide and polynucleotide is one of the most widely accepted models in origin of life research. In this model, the translation system shepherded the RNA World into the extant biology of DNA, RNA, and protein. Here, we examine the RNA World Hypothesis in the context of increasingly detailed information available about the origins, evolution, functions, and mechanisms of the translation system. We conclude that the translation system presents critical challenges to RNA World Hypotheses. Firstly, a timeline of the RNA World is problematic when the ribosome is incorporated. The mechanism of peptidyl transfer of the ribosome appears distinct from evolved enzymes, signaling origins in a chemical rather than biological milieu. Secondly, we have no evidence that the basic biochemical toolset of life is subject to substantive change by Darwinian evolution, as required for the transition from the RNA world to extant biology. Thirdly, we do not see specific evidence for biological takeover of ribozyme function by protein enzymes. Finally, we can find no basis for preservation of the ribosome as ribozyme or the universality of translation, if it were the case that other information transducing ribozymes, such as ribozyme polymerases, were replaced by protein analogs and erased from the phylogenetic record. We suggest that an updated model of the RNA World should address the current state of knowledge of the translation system.

This is a preview of subscription content, access via your institution.

The Ribosome is a Ribozyme: A Look Into the Work of Nobel Prize Winner Thomas Steitz

In the office of Thomas Steitz, Sterling Professor of Molecular Biophysics and Biochemistry and Professor of Chemistry, there are numerous small models of each of the various macromolecular machines that his laboratory has “solved.”

It is an impressive collection that consists of an HIV-1 reverse transcriptase, a set of DNA polymerases in various states along the DNA replication pathway, a pyrrolysyl-tRNA synthetase, and a ribosome.

“My laboratory has been interested in understanding the mechanisms of the molecular machinery involved in the Cen­tral Dogma,” noted Steitz. “We want to make connections between structure and function.”

The Central Dogma of Biology tells us that DNA is transcribed to make a type of RNA, which in turn provides the molecu­lar template to makes proteins, a process termed translation. However, many of the details of that big-picture flow remain to be understood.

During the past several decades, the Steitz laboratory has made considerable advances in elucidating the mechanisms of DNA replication, transcription, and trans­lation. By selecting to study each molecule individually, atomics maps of a variety of macromolecules have been determined.

Many of these advances have led to new rational-based approaches for drug design as well as insight into fundamental pathways in life. Collectively, these results have allowed researchers to understand life at an unprecedented level.

One of Steitz’s most significant achieve­ments came in 2000, when he published a high-resolution structure of the large subunit of the ribosome, the protein synthesis machinery of both prokaryotic and eukaryotic cells. This accomplishment revealed a multitude of details regarding the fundamental macromolecule.

Among these details was the demonstra­tion that the active site of the ribosome is composed solely of ribonucleic acid (RNA). This observation had ground­breaking implications in understanding the molecular evolution of life.

Recently Steitz, along with Venkatraman Ramakrishnan from the United Kingdom and Ada E. Yonath from Israel, was awarded the 2009 Nobel Prize in Chemistry for “studies of the structure and function of the ribosome.” In their press releases, the Nobel Prize Committee notes both the scientific and clinical applications of Steitz’ achievements. The 1.4 million dollar prize was given at the annual awards ceremony in Stockholm, Sweden.

“It is an honor to receive the recogni­tion of the international science commu­nity,” noted Steitz as he stared at a pile of unopened congratulatory letters and cards resting on his office desk.

By employing a powerful technique in structural biophysics, X-ray crystallography, Steitz, Ramakrishnan, and Yonath were able to position each of the atoms of the ribo­some, a molecule that is approximately 2.5 x 106 Daltons in mass and 200 angstroms in diameter. The three researchers pub­lished the crystal structure independently of one other.

X-ray crystallography is one of the most fundamental yet powerful techniques for solving the structure of macromolecules. At present, it is the one possible way of studying the position of individual atoms.

In X-ray crystallography, researchers purify a macromolecule using a variety of column chromatography techniques (such as gel-filtration or ion-exchange). Once a sample is sufficiently pure, it is added in various concentrations to a variety of buffer solutions in an attempt to order the molecules and form well-defined protein crystals. This particular process is labori­ous and, in many circumstances, may take years to perfect.

Once a crystal is formed, it is taken to a particular type of particle accelerator known as a synchrotron, where it is bom­barded with high-energy X-rays. The X-rays hit the atoms within the crystal and produce a diffraction pattern, which represents the positions of individual atoms. After analysis of a multitude of diffraction patterns, a final structure is modeled using crystallog­raphy software and rigorously examined to ensure that it accurately represents the in vivo structure.

Using a process similar to that outlined above, Steitz was able to solve the structure of the ribosome. However, given the size and complexity of the ribosome, numerous technical difficulties were presented, which Steitz had to overcome before completely solving the structure.

Peter Moore, Sterling Professor of Chemistry, Professor of Molecular Bio­physics and Biochemistry, and a long-time collaborator and friend of Steitz, notes that once ribosome crystals were formed, it was relatively simple to measure the diffraction pattern of the macromolecule. However, determining the relative phases of each of the thousands of reflections contained was a difficult task.

The “phase problem” as it has been called in X-ray crystallography is a loss of phase information when taking physical measurements. The light detectors that are used in diffraction experiments collect only the intensity of the light. Since wavelengths contain both amplitudeand phase a crucial piece of information is lost. This informa­tion must be reconstituted to effectively determine the electron density map of a macromolecule, in this case the ribosome.

To circumvent the phase challenge, Steitz modified and then applied an approach Max Perutz devised to solve structures of macromolecules. He utilized a large multi-atom tungsten cluster to serve as a “reference point.” This cluster would bind with the ribosome and diffract as a distinct reference spot. This reference information allowed Steitz to form a complete data once multiple partial sets were integrated.

An additional problem rests within the limitations of computing software. As Steitz states, “The computer technology used in solving the ribosome was not available until the 1990s. It would have been impossible to accomplish the task before then.”

Overall, Steitz had to employ familiar techniques in a novel fashion.” Once these problems were solved, solving the structure of the ribosome was relatively straight-forward,” noted Steitz. Utilizing the complete diffraction data, which contained the phase information, Steitz performed a Fourier synthesis to determine the electron density of the ribosome and subsequently, its structure.

Co-crystal structure of an antibiotic bound to the large ribosomal subunit. Image courtesy of Thomas Steitz.

Today the Steitz laboratory is tackling a plethora of new questions in molecu­lar biology. For instance, his laboratory has recently solved many structures of the ribosome complexed with a variety of antibiotics to provide a textbook for ribosome-targeted, rational-based drug design. Already, these structural insights are being successfully employed at Rib-X Pharmaceuticals, a company both Steitz and Moore help establish in New Haven, to design a new generation of effective anti­biotics. Furthermore, Steitz is investigating the various states of the ribosome during the translation pathway. These studies have the potential for profound effects in medicine and pharmaceuticals.

Peter Moore remarked, “By understand­ing how various antibiotics target the ribo­some, you can learn how to develop entire new classes of antibiotics.”

The structure of the ribosome for Steitz is not the conclusion of a journey, but the beginning of an exciting one. “For every answered question,” he said, “ten more emerge. No matter how much one pathway or process is studied, there will always be questions left unanswered.”

About the Author
PHONG LEE is a junior in Branford College majoring in Molecular Biophysics and Bio­chemistry. He is currently working in Professor Yorgo E. Modis’s laboratory studying both the structural mechanism of innate immunity and the membrane fusion of the Hepatitis-G virus.

The author would like to thank Professor Thomas Steitz, Professor Peter Moore, and Ms. Peggy A. Eatherton for their assistance with the article.

Access options

Get full journal access for 1 year

All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.

Get time limited or full article access on ReadCube.

All prices are NET prices.

Ribosome the ultimate protein synthesizing nano machine

23nm, 1nm=10 -9 M) and complexity in its function ie synthesizing thousands of proteins required for the cell, is not easy to comprehend. I think, I don’t have the knowledge to site any known machine in size or complexity as marvellous as ribosomes and that is it.

15000, ribosomes, making up 25% of the dry weight of the cell. In prokaryotes, ribosomes are smaller and made up of two unequal subunits 30S and 50S (Svedberg unit) and combined to form a sedimentation co-efficient of 70S (18nm diameter). Whereas, Eukaryotic ribosomes are bigger (23nm) and more complex, 80S and made up of subunits 40S and 60S.

100). Electron microscopic study revealed tandem repeats of rRNA genes (rDNA) are situated along the DNA molecule. The selective amplification of rDNA is necessary for production of large number of ribosomes that are required for fertilized egg to synthesize enough proteins for embryonic development. The transcription of rDNA is mediated by RNA polymerase I (an RNA pol I for every 100 bp of DNA). The rRNA precursor is further processed by RNA and proteins to form final rRNA product. Adjacent rDNA genes are separated by spacer DNA which is not transcribed.

Back to the future of RNA structure

The first issue of RNA appeared 20 years ago in March, 1995 with Tim Nilsen as Editor, with a cover that featured a ribbon diagram of a model of the self-splicing Group I intron based on phylogenetic analysis by Francois Michel and Eric Westhof. At that time, there were fewer than 15 RNA structures, including RNA-DNA and RNA-protein complexes, in the Brookhaven Data Base (now known as the Protein Data Base). By the end of 1996, there were over 40 this 𠇎xplosive” growth in RNA structural studies led to the first ever meeting on RNA Structure, organized by Harry Noller and others and held at UC Santa Cruz in June, 1997. Olke Uhlenbeck was recruited to write a review of that meeting, and he in turn recruited Art Pardi and myself to help him (Uhlenbeck, Pardi, and Feigon, Cell, 1997). Looking back, it was an exciting time! The review of the meeting provides a window into that time frame. The structure of the Tetrahymena group I intron P4/P6 domain had just been published (Cate et al Doudna, Nature, 1996), which, at 160 nt, was the largest RNA structure solved since the 96 nt phenylalanine tRNA structures reported 22 years earlier independently by the Aaron Klug and Alex Rich labs. (The P4/P6 structure became the second cover picture of RNA, volumes 3𠄶 from 1997�.) This structure verified many structural predictions based on biochemical data while also providing atomic details of helical structure, folding, packing, and interactions, many of which could not have been predicted. About 33 other RNA (only) solution NMR (23) and X-ray crystal (10) structures were presented in talks and posters at the meeting (about half of which were not yet published). These comprised domains of rRNA (the largest being 62 nt of 5S rRNA), viral RNAs, ribozymes, mRNA, and snRNAs the hammerhead ribozyme in vitro selected RNA aptamers and RNA duplexes. On a personal note, our first structure of an RNA aptamer, the ATP aptamer bound to AMP, was published in RNA (Dieckman et al. 1996), with an accompanying perspective on NMR structures of aptamers by Tom Cech and Alexander Szewczak. Each of these RNA structures revealed details of new and/or common RNA structure motifs, including U-turns, A-platforms, reverse sugars, inter-strand purine stacks, base zippers, base triples, base quadruples, and pseudoknots, irregular helices with non-Watson-Crick base pairs, bulges, internal loops and tetraloops, along with details of the long range tetraloop receptor-tetraloop interactions. Major themes of the 1997 meeting, in addition to structures of small RNA motifs, were roles of monovalent and divalent cations on folding, stability, and catalysis including the hammerhead ribozyme, biochemical and computational methods for determining RNA folding and dynamics, structures of RNA-protein complexes, and predictions of large RNA structures (i.e., group II intron, RNAse P RNA, rRNA) based on phylogenetic, thermodynamic, cross-linking, chemical probing, and other data. Of the RNA-protein complexes were structures of tRNA bound to tRNA synthetases and to elongation facture Tu, but only a few examples of other RNA-protein complexes. These included crystal structures of a complex of virus MS2 coat protein bound to a hairpin RNA, the first dsRBD-RNA complex by Steve Schultze, U2A-U2B′′RRM-RNA ternary complex by Kiyoshi Nagai, and the first solution NMR structure of an RNA-protein complex, the U1A N-terminal RRM with U1A mRNA fragment from Gabriele Varani's lab presented by his former graduate student and my then current postdoc Frederic Allain. Nearly 20 years ago the ribosome was considered “the ultimate example of a big problem in RNA structural biology.” Pictures of ribosome crystals were shown by Harry Noller and Tom Steitz, but solving X-ray structures of the ribosome still appeared to be a monumental challenge.

Despite our predictions to the contrary, the first high-resolution structures of the ribosome were published before the millennium (and the 16S rRNA structure in the 70S ribosome was on the cover of RNA for volumes 7 and 8, years 2001�). Today we have many structures of ribosomal small and large subunits, complete ribosomes, and ribosomes with different subunits, at various stages of translation, and with inhibitors. While still a challenge, many crystal structures of complete ribozymes as well as riboswitches have been determined. A current search for RNA in the PDB yielded 1068 structure hits, of which 248 were published in RNA (and since 2003 each issue of RNA has featured a different structure on the cover).

When the RNA journal began and the first RNA Structure meeting was held, it appeared evident that RNA structural biology had reached a new frontier. Yet as that frontier has receded, new frontiers have come into view. While the major themes of the first RNA Structure meeting would be familiar to readers of RNA in a meeting held in 2015, the field of RNA molecular and structural biology has expanded enormously. Twenty years ago the known important RNAs were tRNA, mRNA, rRNA, snRNAs, ribozymes, introns, viral RNAs (pseudoknots), and in vitro selected aptamers. Small nucleolar RNAs were just starting to be characterized and 7SK RNA was an exception to the rule. Even the most RNA-centric “RNA world” scientist could not have predicted the prevalence and variety of biologically important RNAs and RNPs and their myriad functions that have now been discovered. A recent elegant review by Tom Cech and Joan Steitz (Cell, 2014) lists 54 classes of RNA, including many with vast numbers such as riboswitches, lncRNAs, microRNAs, snoRNAs and scaRNAs. Many of these non-coding RNAs function as a component of RNPs, and many of the RNPs are dynamic assemblies with proteins that come on and off a core RNP for regulation of function. Many RNAs also assume different structures for different functions, e.g., viral RNAs as well as riboswitches. The growing numbers of functional non-coding RNA and RNPs present new targets as well as new challenges for RNA structural biology.

Fortunately, the last few years have presented structural biologists with new and improved tools for structural biology. Once crystals are obtained, data collection at synchrotrons can be fast and efficient. New NMR methods for RNA structure determination, for investigating RNA dynamics over a wide range of time scales, and for detecting small populations (invisible states) of RNA conformations have been developed, and NMR is also one of the best tools for detecting weak interactions. Small angle X-ray scattering (SAXS) has emerged as a useful tool to determine overall shapes of RNAs and RNPs, and is particularly useful when used in combination with high-resolution structures of domains. Fluorescence resonance energy transfer (FRET) and especially single molecule FRET (smFRET) have been used to decipher conformational changes in riboswitches, the ribosome, telomerase, and more. Negative stain and cryolectron microscopy has been and will continue to be tremendously useful for studying RNA assemblies even when only lower resolution EM maps are obtained, as subunits can be localized by affinity labeling and the maps can be fit with higher resolution crystal and NMR structures of domains. This was the case for the EM structure of Tetrahymena telomerase, a collaboration between the laboratories of Kathy Collins, Hong Zhou, and myself (Jiang et al., Zhou, Collins, Feigon, Nature, 2013). But perhaps nothing is more significant than the amazing and ongoing progress in cryoEM single particle analysis over the past couple of years (Bai, McMullan, Scheres, TIBS, 2015). The recent development of direct-electron detectors and improved image processing have together led to determination of EM structures at resolutions better than 3.5 Å. Notable among these for the RNA field are several structures of ribosomes including the recently reported structure of the human mitochondrial ribosome large subunit. Some major advantages of cryoEM are the very small amounts of sample needed (as little as 0.1 mg), no requirement for crystallization (for X-ray structures), and in favorable cases the ability to sort out sample impurities and structural heterogeneity during image classification. Although still a considerable challenge, it is also clear that near atomic resolution EM structures are not necessarily limited to large assemblies like the ribosome. A very recent 2.9 Å EM structure of the 440 kD anthrax pore assembly provides an example. Applications of cryoEM to RNA and RNPs other than the ribosome using the new hardware and software are only just beginning, and will clearly bring many exciting new results in the years to come. There are a number of challenges unique to RNA that will need to be overcome, including RNA inter-domain flexibility. A hybrid structural biology approach, incorporating data from NMR, X-ray crystallography, and low and high resolution EM will provide a path to determine not only structures but also dynamics of RNA and RNA assemblies. Coupled with biochemistry these studies should provide new information on RNA and RNP catalytic mechanisms, interactions, and assembly. It is clear that we are entering a new frontier of RNA structural biology. It's (still) an exciting time!

While much has changed in the last 20 years in the RNA world, one constant has been Tim Nilsen as Editor of RNA. I would like to take this opportunity to thank him for his profound knowledge, insights and influence on the RNA field, which have benefited RNA scientists both young and old.

Watch the video: #Animation #Ribozyme Structure and Activity #Molecular Biology of the Gene #SD LIFE SCIENCE (January 2022).