Ribosomal subunit

50S ribosomal subunits. From Yonath et al. (1990), with permission. 

Characterized, Three-Dimensional Crystals of Ribosomal Particles 

Source0 Growth form4 Cell dimensions (A) symmetry Resolution (A) 

Recently von Boehlen et al. (1991) have obtained an improved crystal form of the large (50S) ribosomal subunit of H. marismortui, which diffracts to 3 A resolution, by the addition of 1 mAf Cd2+ to a crystallization medium that contained over 1.9 M of other salts. These improved crystals are isomorphous with the previously reported ones, and, as was the case for the previous crystals, they show no measurable decay after a few days of synchrotron irradiation at cryotemperatures. The new crystals are of adequate mechanical strength. Initial phasing studies by specific and quantitative deriva-tization with super-dense heavy-atom clusters and by real- and reciprocal-space rotation searches are in progress. 

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It has been known for some time that tetracyclines are accumulated by bacteria and prevent bacterial protein synthesis (Fig. 4). Furthermore, inhibition of protein synthesis is responsible for the bacteriostatic effect (85). Inhibition of protein synthesis results primarily from dismption of codon-anticodon interaction between tRNA and mRNA so that binding of aminoacyl-tRNA to the ribosomal acceptor (A) site is prevented (85). The precise mechanism is not understood. However, inhibition is likely to result from interaction of the tetracyclines with the 30S ribosomal subunit because these antibiotics are known to bind strongly to a single site on the 30S subunit (85). 

Protein-Free 50S Ribosomal Subunits Catalyze Peptide Bond Formation In Vitro... 

Perhaps the most significant case of catalysis by RNA occurs in protein synthesis. Harry F. NoIIer and his colleagues have found that the peptidyl transferase reaction, which is the reaction of peptide bond formation during protein synthesis (Figure 14.24), can be catalyzed by 50S ribosomal subunits (see Chapter 12) from which virtually ail of the protein has been removed. These... 

Other examples include rifampin resistance due to mutations in the ipoB gene encoding the (3-subunit of RNA polymerase, or oxazolidinone resistance due to a G2576T mutation in the gene for the 23 S rRNA as central part of the 50S large ribosomal subunit. Macrolide resistance is based upon the alteration of nucleotide A2058 by a point mutation. 

Macrolides are a group of antibiotics, produced in nature by many actinomycetes strains, that are composed of a 12- to 16-membered lactone ring, to which one or more sugar substituents is attached. They target the peptidyl transferase center on the 50S ribosomal subunit and function primarily by interfering with movement of the nascent peptide away from the active site and into the exit tunnel. 

Macrolide, lincosamide and streptogramin B resistance (MLSb phenotype) can be linked to specific nucleotide changes within the 23 S rRNA of the large ribosomal subunit, mainly at position A2058 or neighbouring bases (E. coli numbering). This is the... 

Oxazolidinones are a new class of synthetic antimicrobial agents, which have activity against many important pathogens, including methicillin-resistant Staphylococcus aureus and others. Oxazolidinones (e.g. linezolid or eperezolid) inhibit bacterial protein synthesis by inhibiting the formation of the 70S initiation complex by binding to the 50S ribosomal subunit close to the interface with the 3OS subunit. 

Figure 1 Schematic drawing of the morphology of the ribosome. The ribosomal subunits are labeled, as are the approximate locations of their respective functional centers. The drawing is a transparent view from the solvent side of the small subunit. Transfer RNAs are shown in different binding states with the arrow indicating their direction of movement through the ribosome. The tRNA anticodon ends are oriented towards the viewer, whereas the 3-ends of the tRNAs are oriented towards the peptidyl transferase region on the large subunit. The letters h and b denote the head and body regions on the 30S subunit, respectively.

Carter AP, Clemons WM, Brodersen DE et al (2000) Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407 340-348... 

Brodersen DE, Clemons WM, Carter AP et al (2000) The structural basis for the action of the antibiotics tetracycline, pactamycin and hygromycin B on the 30S ribosomal subunit. Cell 103 1143-1154... 

The ribosomal subunits are defined according to their sedimentation velocity in Svedberg units (40S or 60S). This table illustrates the total mass (MW) of each. The number of unique proteins and their total mass (MW) and the RNA components of each subunit in size (Svedberg units), mass, and number of bases are listed. 

Initiation of protein synthesis requires that an mRNA molecule be selected for translation by a ribosome. Once the mRNA binds to the ribosome, the latter finds the correct reading frame on the mRNA, and translation begins. This process involves tRNA, rRNA, mRNA, and at least ten eukaryotic initiation factors (elFs), some of which have multiple (three to eight) subunits. Also involved are GTP, ATP, and amino acids. Initiation can be divided into four steps (1) dissociation of the ribosome into its 40S and 60S subunits (2) binding of a ternary complex consisting of met-tRNAf GTP, and eIF-2 to the 40S ribosome to form a preinitiation complex (3) binding of mRNA to the 40S preinitiation complex to form a 43S initiation complex and (4) combination of the 43S initiation complex with the 60S ribosomal subunit to form the SOS initiation complex. 

Two initiation factors, eIF-3 and elF-lA, bind to the newly dissociated 40S ribosomal subunit. This delays its reassociation with the 60S subunit and allows other translation initiation factors to associate with the 40S subunit. 

The first step in this process involves the binding of GTP by eIF-2. This binary complex then binds to met-tRNAf a tRNA specifically involved in binding to the initiation codon AUG. (There are two tRNAs for methionine. One specifies methionine for the initiator codon, the other for internal methionines. Each has a unique nucleotide sequence.) This ternary complex binds to the 40S ribosomal subunit to form the 43S preinitiation complex, which is stabilized by association with eIF-3 and elF-lA. 

Biochemical and genetic experiments in yeast have revealed that the b poly(A) tail and its binding protein, Pablp, are required for efficient initiation of protein synthesis. Further studies showed that the poly(A) tail stimulates recruitment of the 40S ribosomal subunit to the mRNA through a complex set of interactions. Pablp, bound to the poly(A) tail, interacts with eIF-4G, which in turn binds to eIF-4E that is bound to the cap structure. It is possible that a circular structure is formed and that this helps direct the 40S ribosomal subunit to the b end of the mRNA. This helps explain how the cap and poly(A) tail structures have a synergistic effect on protein synthesis. It appears that a similar mechanism is at work in mammalian cells. 

The binding of the 60S ribosomal subunit to the 48S initiation complex involves hydtolysis of the GTP bound to elF-2 by elF-5. This teaction tesults in telease of the initiation factots bound to the 48S initiation complex (these factots then ate tecycled) and the tapid association of the 40S and 60S subunits to fotm the 80S ribosome. At this point, the met-tRNA is on the P site of the ribosome, ready for the elongation cycle to commence. 

The 4F complex is particularly important in controUing the rate of protein translation. As described above, 4F is a complex consisting of 4E, which binds to the m G cap strucmre at the 5 end of the mRNA, and 4G, which serves as a scaffolding protein. In addition to binding 4E, 4G binds to elF-3, which hnlcs the complex to the 40S ribosomal subunit. It also binds 4A and 4B, the ATPase-hehcase complex that helps unwind the RNA (Figure 38—7). 

The a-amino group of the new aminoacyl-tRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA occupying the P site (peptidyl or polypeptide site). At initiation, this site is occupied by aminoacyl-tRNA mef. This reaction is catalyzed by a peptidyltransferase, a component of the 285 RNA of the 605 ribosomal subunit. This is another example of ribozyme activity and indicates an important—and previously unsuspected—direct role for RNA in protein synthesis (Table 38-3). Because the amino acid on the aminoacyl-tRNA is already activated, no further energy source is required for this reaction. The reaction results in attachment of the growing peptide chain to the tRNA in the A site. 

The now deacylated tRNA is attached by its anticodon to the P site at one end and by the open GGA tail to an exit (E) site on the large ribosomal subunit (Figure 38-8). At this point, elongation factor 2 (EE2) binds to and displaces the peptidyl tRNA from the A site to the P site. In turn, the deacylated tRNA is on the E site, from which it leaves the ribosome. The EF2-GTP complex is hydrolyzed to EF2-GDP, effectively moving the mRNA forward by one codon and leaving the A site open for occupancy by another ternary complex of amino acid tRNA-EFlA-GTP and another cycle of elongation. 

Figure 38-10. Picornavimses disrupt the 4F complex. The 4E-4G complex (4F) directs the 40S ribosomal subunit to the typical capped mRNA (see text). 4G alone is sufficient for targeting the 40S subunit to the internal ribosomal entry site (IRES) of viral mRNAs. To gain selective advantage, certain viruses (eg, poliovirus) have a protease that cleaves the 4E binding site from the amino terminal end of 4G. This truncated 4G can direct the 40S ribosomal subunit to mRNAs that have an IRES but not to those that have a cap. The widths of the arrows indicate the rate of translation initiation from the AUG codon in each example.

Other antibiotics inhibit protein synthesis on all ribosomes (puromycin) or only on those of eukaryotic cells (cycloheximide). Puromycin (Figure 38—11) is a structural analog of tyrosinyl-tRNA. Puromycin is incorporated via the A site on the ribosome into the carboxyl terminal position of a peptide but causes the premature release of the polypeptide. Puromycin, as a tyrosinyl-tRNA analog, effectively inhibits protein synthesis in both prokaryotes and eukaryotes. Cycloheximide inhibits peptidyltransferase in the 60S ribosomal subunit in eukaryotes, presumably by binding to an rRNA component. 

These macromolecules include histones, ribosomal proteins and ribosomal subunits, ttansctiption factors, and mRNA molecules. The transport is bidirectional and occurs through the nucleat pote complexes (NPCs). These are complex stmctures with a mass approximately 30 times that of a ribosome and are composed of about 100 diffetent proteins. The diameter of an NPC is approximately 9 run but can increase up to ap-ptoximately 28 nm. Molecules smaller than about 40 kDa can pass through the channel of the NPC by diffusion, but special translocation mechanisms exist fot latget molecules. These mechanisms are under intensive investigation, but some important features have already emerged. 

Bacterial ribosome function Aminoglycosides Tetracyclines Chloramphenicol Macrolides, azalides Fusidic acid Mupirocin Distort SOS ribosomal subunit Block SOS ribosomal subunit Inhibits peptidyl transferase Block translocation Inhibits elongation factor Inhibits isoleucyl-tRNA synthesis No action on 40S subunit Excluded by mammalian cells No action on mammalian equivalent No action on mammalian equivalent Excluded by mammalian cells No action on mammalian equivalent... 

These agents bind selectively to a region of the SOS ribosomal subunit close to that of chloramphenicol and erythromycin. They block elongation of the peptide chain by inhibition of peptidyl transferase. 

Alteration in binding site Streptomycin Protein S12 component of 30S ribosomal subunit determines sensitivity or resistance... 

Plasmid- or transposon-encoded ribosomal protection factors are a second mechanism of resistance to the tetracyclines. These proteins are believed to alter the tetracycline binding site on the 308 ribosomal subunit, lowering the affinity for the drugs. 

Plasmid- or transposon-mediated resistance common to the MLS group is due to RNA methylase genes emiA, emiB and ermC) which code for the methylation of an adenine residue in 23 S rRNA. Methylation prevents the drugs from binding to the 508 ribosomal subunit and confers resistance to all MLS antibiotics. 

Lee, S.W., Berger, S.J., Martinovic, S., Pasa-Tolic, L., Anderson, G.A., Shen, Y., Zhao, R., Smith, R.D. (2002). Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR. Proc. Natl. Acad. Sci. USA 99, 5942-5947. 

The ribosome is a ribozyme this is how Cech (2000) commented on the report by Nissen et al. (2000) in Science on the successful proof of ribozyme action in the formation of the peptide bond at the ribosome. It has been known for more than 30 years that in the living cell, the peptidyl transferase activity of the ribosome is responsible for the formation of the peptide bond. This process, which takes place at the large ribosome subunit, is the most important reaction of protein biosynthesis. The determination of the molecular mechanism required more than 20 years of intensive work in several research laboratories. The key components in the ribosomes of all life forms on Earth are almost the same. It thus seems justified to assume that protein synthesis in a (still unknown) common ancestor of all living systems was catalysed by a similarly structured unit. For example, in the case of the bacterium E. coli, the two subunits which form the ribosome consist of 3 rRNA strands and 57 polypeptides. Until the beginning of the 1980s it was considered certain that the formation of the peptide bond at the ribozyme could only be carried out by ri-bosomal proteins. However, doubts were expressed soon after the discovery of the ribozymes, and the possibility of the participation of ribozymes in peptide formation was discussed. 

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