30.5 What Are the Mechanics of mRNA Translation? 973 GTP Hydrolysis Fuels the Conformational Changes That Drive Ribosomal Functions Two GTPs are hydrolyzed for each amino acid residue incorporated into peptide during chain elongation: one upon EF-Tu–mediated binding of aa-tRNA and one more in translocation. The role of GTP (with EF-Tu as well as EF-G) is mechani- cal, in analogy with the role of ATP in driving muscle contraction (see Chapter 16). GTP binding induces conformational changes in ribosomal components that actively engage these components in the mechanics of protein synthesis; subse- quent GTP hydrolysis followed by GDP and Pi release relax the system back to the initial conformational state so that another turn in the cycle can take place. The energy expenditure for protein synthesis is at least four high-energy phosphoric anhydride bonds per amino acid. In addition to the two provided by GTP, two from ATP are expended in amino acid activation via aminoacyl-tRNA synthesis (see Figure 30.3). A DEEPER LOOK Molecular Mimicry—The Structures of EF-TuϺA minoacyl-tRNA, EF-G, and RF-3 EF-Tu and EF-G compete for binding to ribosomes. EF-Tu has the unique capacity to recognize and bind any aminoacyl-tRNA and de- liver it to the ribosome in a GTP-dependent reaction. EF-G catalyzes GTP-dependent translocation. The structure of the EF-TuϺtRNA complex is remarkably similar to the structure of EF-G (EF-TuϺ tRNA is shown on the left in the figure; EF-G is in the center). The EF-TuϺtRNA structure is Thermus aquaticus EF-TuϺPhe-tRNA Phe com- plexed with GMPCP (purple); GMPCP is a nonhydrolyzable analog of GTP (pdb id ϭ 1TTT). The EF-G structure has GDP bound (pur- ple) (pdb id ϭ 1DAR). Note that parts of the EF-G structure mimic the structure of the tRNA molecule. Both EF-Tu and EF-G bind to the factor-binding center, which is located on the L7/L12 side of 50S ribosomal subunit. Part of this center has an associated GTPase function that plays an integral role in the binding and release of these factors. Nature has extended this mimicry with RF-3, a ribosome- binding protein essential to the termination phase of protein syn- thesis. RF-3 is a GTP-binding protein that looks like a tRNA (the structure of RF-3 with bound GDP (purple) is shown on the right in the figure) (pdb id ϭ 2H5E). One view of early evolution suggests that RNA was the primor- dial macromolecule, fulfilling all biological functions, including those of catalysis and information storage that are now assumed for the most part by proteins and DNA. The mimicry of EF-TuϺ tRNA by EF-G may represent a fossil of early macromolecular evo- lution when the proteins first began to take over some functions of RNA by mimicking shapes known to work as RNAs. ᮣ EF-TuϺtRNA (left); EF-G (center); and RF-3 (right). 974 Chapter 30 Protein Synthesis Peptide Chain Termination Requires a G-Protein Family Member The elongation cycle of polypeptide synthesis continues until the 70S ribosome en- counters a “stop” codon. At this point, polypeptidyl-tRNA occupies the P site and the arrival of a “stop” or nonsense codon in the A site signals that the end of the polypep- tide chain has been reached (Figure 30.24) These nonsense codons are not “read” by any “terminator tRNAs” but instead are recognized by specific proteins known as release factors, so named because they promote polypeptide release from the ribo- some. The release factors bind at the A site. RF-1 recognizes UAA and UAG, whereas RF-2 recognizes UAA and UGA. RF-1 and RF-2 are members of the guanine nu- cleotide exchange factor (GEF) family of proteins, which includes EF-Ts. Like EF-G, RF-1 and RF-2 interact well with the ribosomal A-site structure. Furthermore, these release factors “read” the nonsense codons through specific tripeptide sequences that serve as the RF protein equivalent of the tRNA anticodon loop. There is about one molecule each of RF-1 and RF-2 per 50 ribosomes. Ribosomal binding of RF-1 or RF-2 is competitive with EF-G. RF-1 or RF-2 recruit a third release factor, RF-3, complexed with GTP; this protein is a structural mimic of tRNA. RF-3 is the fourth G-protein family member (the other three are IF-2, EF-Tu, and EF-G) involved in protein synthesis. All share the same ribosomal binding site. The state of the pep- tidyl-tRNA in the P site determines which is bound and, importantly, the progression of protein synthesis through initiation, elongation, and termination. The presence of release factors with a nonsense codon in the A site creates a 70S ribosomeϺRF-1 (or RF-2)ϺRF-3-GTPϺtermination signal complex that transforms the ribosomal peptidyl transferase into a hydrolase. That is, instead of catalyzing the trans- fer of the polypeptidyl chain from a polypeptidyl-tRNA to an acceptor aminoacyl- tRNA, the peptidyl transferase hydrolyzes the ester bond linking the polypeptidyl chain to its tRNA carrier. Both RF-1 and RF-2 have a conserved GGQ sequence lo- cated in a loop that enters the PTC. This GGQ sequence faces A76 of the P-site tRNA; the closest 23S rRNA residues are A2451 and A2602. RF-1 and RF-2 promote confor- mational adjustments that expose the peptidyl-tRNA ester to attack by a water mole- cule. The conserved Gln in the release factor GGQ motif positions this hydrolytic water molecule through hydrogen-bonding interactions. The peptidyl transferase transfers the polypeptidyl chain to this water molecule instead of an aminoacyl- tRNA. Peptide release is followed by expulsion of RF-1 (or RF-2) from the ribosome (Figure 30.24). This leaves a ribosomeϺmRNAϺP-site tRNA complex that must be dis- assembled by a protein ribosome recycling factor (RRF) with the help of EF-G. The structure of RRF resembles the letter “L”; thus, this protein is also structurally akin to a tRNA. We can now recount the central role played by GTP in protein synthesis. IF-2, EF-Tu, EF-G, and RF-3 are all GTP-binding proteins, and all are part of the G-protein superfamily (whose name is derived from the heterotrimeric G proteins that function in transmembrane signaling pathways, as in Figure 15.19). IF-2, EF-Tu, EF-G, and RF-3 interact with the same site on the 50S subunit, the factor-binding cen- ter, in the 50S cleft. This factor-binding center activates the GTPase activity of these factors, once they become bound. The Ribosomal Subunits Cycle Between 70S Complexes and a Pool of Free Subunits Ribosomal subunits cycle rapidly through protein synthesis. In actively growing bac- teria, 80% of the ribosomes are engaged in protein synthesis at any instant. Once a polypeptide chain is synthesized and the nascent polypeptide chain is released, the 70S ribosome dissociates from the mRNA and separates into free 30S and 50S sub- units (Figure 30.25). Intact 70S ribosomes are inactive in protein synthesis because only free 30S subunits can interact with the initiation factors. Binding of initiation factor IF-3 by 30S subunits and interaction of 30S subunits with 50S subunits are mu- tually exclusive. 30S subunits with bound initiation factors associate with mRNA, but 50S subunit addition requires IF-3 release from the 30S subunit. 30.5 What Are the Mechanics of mRNA Translation? 975 Peptidyl-tRNA A-site empty 5ЈmRNA 3Ј UAAGAUG UAG 5Ј 3Ј UAG GTP RF-1 RF-3 5Ј 3Ј UAG GTP RF-1 RF-3 Uncharged tRNA UAAGAUG UAAGAUG AGC UAC UAA AGC UAC UAA AGC UAC UAA Nascent polypeptide Pro Ala Asn Met Ile Gln Asp Tyr Met His Glu Lys Ser Tyr Met Pro Ala Asn Met Ile Gln Asp Tyr Met His Glu Lys Ser Tyr Met RF-1 RF-3 • GTP + Pro Ala Asn Met Ile Gln Asp Tyr Met His Glu Ser Tyr Lys Met COO – H 2 O Polypeptide GAGUAGAAU 5Ј 3Ј Inactive 70S ribosome AUC AUA C RF-1 RF-3 UAG GDP + P i RRF RRF Glu Glu His His Ala Ala Ile Ile Asn Asn Lys Lys Ser Ser Met Met Ala Asn Met Ile His Glu Ser Lys ACTIVE FIGURE 30.24 The events in peptide chain termination. Test yourself on the con- cepts in this figure at www.cengage.com/login. 976 Chapter 30 Protein Synthesis Polyribosomes Are the Active Structures of Protein Synthesis Active protein-synthesizing units consist of an mRNA with several ribosomes attached to it. Such structures are polyribosomes, or, simply, polysomes (Figure 30.26). All protein synthesis occurs on polysomes. In the polysome, each ribosome is traversing the mRNA and independently translating it into polypeptide. The fur- ther a ribosome has moved along the mRNA, the greater the length of its associ- ated polypeptide product. In prokaryotes, as many as ten ribosomes may be found in a polysome. Ultimately, as many as 300 ribosomes may translate an mRNA, so as many as 300 enzyme molecules may be produced from a single transcript. Eukary- otic polysomes typically contain fewer than 10 ribosomes. 30.6 How Are Proteins Synthesized in Eukaryotic Cells? Eukaryotic mRNAs are characterized by two post-transcriptional modifications: the 5Ј-terminal 7 methyl-GTP cap and the 3Ј-terminal poly(A) tail (Figure 30.27). The 7 methyl-GTP cap is essential for mRNA binding by eukaryotic ribosomes and also enhances the stability of these mRNAs by preventing their degradation by 5Ј-exonucleases. The poly(A) tail enhances both the stability and translational efficiency of eukaryotic mRNAs. The Shine–Dalgarno sequences found at the 5Ј-end of prokaryotic mRNAs are absent in eukaryotic mRNAs. Peptide Chain Initiation in Eukaryotes The events in eukaryotic peptide chain initiation are summarized in Figure 30.28, and the properties of eukaryotic initiation factors, symbolized eIFs, are presented in Table 30.9. As might be expected, eukaryotic protein synthesis is considerably IF-1 IF-1 IF-3 IF-1 IF-3 5Ј 3Ј 5Ј 3Ј Pool of 70S ribosomes Dynamic equilibrium 30S subunit with IF-1 and IF-3 binds mRNA. IF3 blocks 50S subunit binding. IF3 is released before 50S subunit can join. Elongation Termination Free subunits IF-1 IF-3 IF-3 RRF FIGURE 30.25 The ribosome life cycle. Note that IF-3 is released prior to 50S addition. FIGURE 30.26 Electron micrograph of polysomes: multi- ple ribosomes translating the same mRNA. (From Francke, C., et al., 1982. Electron microscopic visualization of a discrete class of giant translation units in salivary gland cells of Chirono- mus tentans.The EMBO Journal 1:59–62. Photo courtesy of Oscar L. Miller, Jr., University of Virginia.) 30.6 How Are Proteins Synthesized in Eukaryotic Cells? 977 more complex than prokaryotic protein synthesis. More than 100 different RNA molecules and 200 proteins are required just for the core translational machinery in Caenorhabditis elegans, a simple animal. Despite such complexity, the overall process is similar to prokaryotic protein synthesis. The eukaryotic initiator tRNA is a unique tRNA functioning only in initiation. Like the prokaryotic initiator tRNA, the eukaryotic version carries only Met. However, unlike prokaryotic f-Met-tRNA i f Met , the Met on this tRNA is not formylated. The eukaryotic initiator tRNA is usually designated tRNA i Met , with the “i” indicating “initiation.” O HN H 2 N N N + N CH 3 OOH CH 2 OP O – O OP O – O OP O – O G 5Ј CH 3 (A) 50–100 OH 3Ј AUG AAUAAA 5Ј Untranslated region Coding region 3Ј Untranslated region Poly(A) tail Polyadenylation signal Initiation codon 7-Methyl GTP “cap” at 5Ј-end O FIGURE 30.27 The characteristic structure of eukaryotic mRNAs. Untranslated regions ranging between 40 and 150 bases in length occur at both the 5Ј- and 3Ј-ends of the mature mRNA. An initiation codon at the 5Ј-end, invariably AUG, signals the translation start site. Met-tRNA i Met • eIF2 • GTP (ternary complex) Met-tRNA i Met 1A 2 40S 60S 1 2 3 eIF2 • GTP 43S preinitiation complex 43S • mRNA complex (48S preinitiation complex) 80S initiation complex 60S subunit eIF-5 eIF2B eIF1 eIF1A eIF3 eIF3 eIF5 GDP GTP 43S40S80S 60S A n mRNA eIF4E eIF4G eIF4A eIF4B PABP Mnk AUG //A n m 7 GTP AUG //A n m 7 GTP m 7 GTP eIF4F eIF2 • GDP 3 1 5 40S 1A 2 3 1 5 40S FIGURE 30.28 The three stages in the initiation of trans- lation in eukaryotic cells. See Table 30.9 for a description of the functions of the eukaryotic initiation factors (eIFs). 978 Chapter 30 Protein Synthesis In particular, initiation of protein synthesis is significantly more complicated in eukaryotes. It can be divided into three fundamental stages: Stage 1: Formation of the 43S preinitiation complex (Figure 30.28, stage 1). Initiation factors eIF1, eIF1A, eIF3, and eIF5 bind to a 40S ribosomal subunit. Then, Met- tRNA i Met (in the form of an eIF2ϺGTPϺMet-tRNA i Met ternary complex) is delivered to the eIF1/1A/3/5Ϻ40S subunit complex. (Unlike in prokaryotes, binding of Met- tRNA i Met by eukaryotic ribosomes occurs in the absence of mRNA, so Met-tRNA i Met binding is not codon-directed.) In mammals, eIF3 is an 800-kD multimer containing at least 13 different subunits. The eIF3 complex binds to the solvent-exposed side of the 40S subunit, away from the face that interacts with mRNA and the 60S subunit. A prominent role of eIF3 is to serve as a platform and scaffold for the recruitment of mRNA and other proteins involved in translation initiation. Stage 2: Formation of the 48S initiation complex (Figure 30.28, stage 2). This stage in- volves binding of the 43S preinitiation complex to mRNA and migration of the 40S ri- bosomal subunit to the correct AUG initiation codon. Binding of mRNA by the 43S preinitiation complex requires a set of proteins including eIF4 group and the (poly)A- binding protein (PABP). Collectively, these proteins recognize the 5Ј-terminal cap and 3Ј-terminal poly(A) tail of an mRNA, unwind any secondary structure in the mRNA, and transfer the mRNA to the 43S preinitiation complex. The eIF4 group includes eIF4B and eIF4F. eIF4F is a trimeric complex consisting of eIF4A (an ATP-dependent RNA helicase), eIF4E (which binds the 5Ј-terminal 7 methyl-GTP of mRNAs), and eIF4G. Because eIF4G interacts with PABP (Figure 30.29), eIF4G serves as the bridge between the cap-binding eIF4E, the poly(A) tail of the mRNA, and the 40S subunit (through interaction with eIF3). These interactions between the 5Ј-terminal 7 methyl- GTP cap and the 3Ј-poly(A) tail initiate scanning of the 40S subunit in search of an AUG codon. eIF4E, the mRNA cap-binding protein of eIF4F, represents a key regulatory ele- ment in eukaryotic translation. eIF4F binding to the cap structure is necessary for association of eIF4B and formation of the 48S preinitiation complex. Translation is inhibited when the eIF4E subunit of eIF4F binds with 4EBP (the eIF4E binding pro- tein). Growth factors stimulate protein synthesis by causing the phosphorylation of 4EBP, which prevents its binding to eIF4E. Factor Subunit Size (kD) Function eIF1 15 Enhances initiation complex formation eIF1A 17 Stabilizes Met-tRNA i binding to 40S ribosomes eIF2 125 GTP-dependent Met-tRNA i binding to 40S ribosomes ␣ 36 Regulated by phosphorylation  50 Binds Met-tRNA i ␥ 55 Binds GTP, Met-tRNA i eIF2B 270 Promotes guanine nucleotide exchange on eIF2 eIF2C 94 Stabilizes ternary complex in presence of RNA eIF3 800 Promotes Met-tRNA i and mRNA binding eIF4F 243 Binds to mRNA caps and poly(A) tails; consists of eIF4A, eIF4E, and eIF4G; RNA helicase activity unwinds mRNA 2° structure eIF4A 46 Binds RNA; ATP-dependent RNA helicase; promotes mRNA binding to 40S ribosomes eIF4E 24 Binds to 5Ј-terminal 7 methyl-GTP cap on mRNA eIF4G 173 Binds to PABP eIF4B 80 Binds mRNA; promotes RNA helicase activity and mRNA binding to 40S ribosomes eIF5 49 Promotes GTPase of eIF2, ejection of eIF2 and eIF3 eIF5B 175 Ribosome-dependent GTPase activity; mediates 40S and 60S joining eIF6 Dissociates 80S; binds to 60S TABLE 30.9 Properties of Eukaryotic Translation Initiation Factors 30.6 How Are Proteins Synthesized in Eukaryotic Cells? 979 Stage 3: Formation of the 80S initiation complex. When the 48S preinitiation com- plex stops at an AUG codon, GTP hydrolysis in the eIF2ϺMet-tRNA i Met ternary com- plex causes ejection of the initiation factors bound to the 40S ribosomal subunit. EIF5, in conjunction with eIF5B, acts here by stimulating the GTPase activity of eIF2. Ejection of the eIFs is followed by 60S subunit association to form the 80S ini- tiation complex, whereupon translation begins (Figure 30.28, stage 3). eIF2ϺGDP is recycled to eIF2:GTP by eIF2B (eIF2B is a guanine nucleotide exchange factor). Control of Eukaryotic Peptide Chain Initiation Is One Mechanism for Post-Transcriptional Regulation of Gene Expression Regulation of gene expression can be exerted post-transcriptionally through con- trol of mRNA translation. Phosphorylation/dephosphorylation of translational components is a dominant mechanism for control of protein synthesis. Several ini- tiation factors—eIF2␣, eIF2B, eIF4E, eIF4G, 40S ribosomal protein S6, and two eu- karyotic elongation factors, eEF1 and eEF2 (see following)—have been identified as targets of regulatory controls. Modification of some factors affects the rate of mRNA translation; modification of others affects which mRNAs are selected for translation. Peptide chain initiation, the initial phase of the synthetic process, is the optimal place for such control. Phosphorylation of S6 facilitates initiation of protein synthesis, resulting in a shift of the ribosomal population from inactive ri- bosomes to actively translating polysomes. S6 phosphorylation is stimulated by serum growth factors (see Chapter 32). On the other hand, the phosphorylation of some translational components inhibits protein synthesis. For example, the ␣-subunit of eIF2 can be reversibly phosphorylated at a specific Ser residue by an eIF2␣ kinase/phosphatase system (Figure 30.30). Four different eIF2␣ kinases are known; each is responsive to specific metabolic signals. Phosphorylation of eIF2␣ inhibits peptide chain initiation. Phosphorylation of eIF2 by heme-regulated inhibitor (HRI, the heme-inhibited eIF2␣ kinase) is an important control govern- ing globin synthesis in reticulocytes. If heme for hemoglobin synthesis becomes limiting in these cells, eIF2␣ is phosphorylated, so globin mRNA is not translated and chains are not synthesized. Availability of heme inhibits HRI, leading to re- sumption of protein synthesis upon phosphatase-mediated removal of the phos- phate group from the Ser residue. 40S 2 Met i -tRNA 1A Mnk N-term PABP AAAAAAAAAAAA PABP 5 4A 4E 4B 4A 3 1 AGU A U A 4G FIGURE 30.29 The 48S initiation complex. mRNA is shown as a black line, its 5Ј-cap as a black circle, and its coding region as a yellow bar beginning with AUG and ending with UAA; downstream lies the poly(A) tail. The initiator Met-tRNA i is shown as a pitchfork bound to the AUG initiation codon.The various initiation factors are shown by their numerical and letter codes. Mnk is a stress- and mitogen-activated protein kinase that phos- phorylates eIF4E, increasing its affinity for the 5Ј-cap. (Adapted from Figure 2 in Rhoads, R., Dinkova,T.D., and Korneeva, N. L., 2006. Mechanism and regulation of translation in C. elegans.WormBook 28:1–18.) 980 Chapter 30 Protein Synthesis eIF2 ␣ P  ␥ eIF2 H 2 O ␣  ␥ P eIF2B P eIF2B ␣  ␥ Tight complex ADP ATP eIF2␣ kinase Phosphoprotein phosphatase FIGURE 30.30 Control of eIF2 functions through reversible phosphorylation of a Ser residue on its ␣-subunit. The phosphorylated form of eIF2 (eIF2–P) enters a tight complex with eIF2B and is unavailable for initiation. HUMAN BIOCHEMISTRY Diphtheria Toxin ADP-Ribosylates eEF2 Diphtheria arises from infection by Corynebacterium diphtheriae bacte- ria carrying bacteriophage corynephage . Diphtheria toxin is a phage-encoded enzyme secreted by these bacteria that is capable of inactivating a number of GTP-dependent enzymes through covalent attachment of an ADP-ribosyl moiety derived from NAD ϩ . That is, diphtheria toxin is an NAD ϩ -dependent ADP-ribosylase. One target of diphtheria toxin is the eukaryotic translocation factor, eEF2. This protein has a modified His residue known as diphthamide. Diph- thamide is generated post-translationally on eEF2; its biological func- tion is unknown. (EF-G of prokaryotes lacks this unusual modifica- tion and is not susceptible to diphtheria toxin.) Diphtheria toxin specifically ADP-ribosylates an imidazole-N within the diphthamide moiety of eEF2 (see accompanying figure). ADP-ribosylated eEF2 retains the ability to bind GTP but is unable to function in protein synthesis. Because diphtheria toxin is an enzyme and can act catalyt- ically to modify many molecules of its target protein, just a few mi- crograms suffice to cause death. CH 2 O OH OH O OH OH CH 2 O NH CH 2 N N CHN + CH 3 CH 3 CO CH – OPO O O – O POO N N N N NH 2 (CH 2 ) 2 CH 3 NH 2 C Diphthamide (modified His residue in eEF2) O NH CH 2 N N H CHN + CH 3 CH 3 CO CH (CH 2 ) 2 CH 3 NH 2 C N + H C NH 2 O Nicotinamide Diphtheria toxin – OPO O O CH 2 O – O PO O N + H C NH 2 CH 2 O N OH OH OH OH N N N NH 2 O NAD + ADP-ribosylated diphthamide residue ᮡ Diphtheria toxin catalyzes the NAD ϩ -dependent ADP-ribosylation of selected proteins. ADP-ribosylation of the diphthamide moiety of eukaryotic EF2. (Diphthamide ϭ 2-[3-carboxamido-3-(trimethylammonio)propyl]histidine.) 30.6 How Are Proteins Synthesized in Eukaryotic Cells? 981 Peptide Chain Elongation in Eukaryotes Resembles the Prokaryotic Process Eukaryotic peptide elongation occurs in very similar fashion to the process in prokaryotes. An incoming aminoacyl-tRNA enters the ribosomal A site while pep- tidyl-tRNA occupies the P site. Peptidyl transfer then occurs, followed by transloca- tion of the ribosome one codon further along the mRNA. Two elongation factors, eEF1 and eEF2, mediate the elongation steps. eEF1 consists of two components: eEF1A and eEF1B. eEF1A is the eukaryotic counterpart of EF-Tu; it serves as the aminoacyl-tRNA binding factor and requires GTP. eEF1B is the eukaryotic equivalent of prokaryotic EF-Ts; it catalyzes the exchange of bound GDP on eEF1ϺGDP for GTP so that active eEF1ϺGTP can be regenerated. EF2 is the eukaryotic translocation fac- tor. eEF2 (like its prokaryotic kin, EF-G) binds GTP, and GTP hydrolysis accompa- nies translocation. Eukaryotic Peptide Chain Termination Requires Just One Release Factor Whereas prokaryotic termination involves three different release factors (RFs), just one RF is sufficient for eukaryotic termination. Eukaryotic RF binding to the ribo- somal A site is GTP-dependent, and RFϺGTP binds at this site when it is occupied by a termination codon. Then, hydrolysis of the peptidyl-tRNA ester bond, hydroly- sis of GTP, release of nascent polypeptide and deacylated tRNA, and ribosome dis- sociation from mRNA ensue. Inhibitors of Protein Synthesis Protein synthesis inhibitors have served two major, and perhaps complementary, pur- poses. First, they have been very useful scientifically in elucidating the biochemical mechanisms of protein synthesis. Second, some of these inhibitors affect prokaryotic but not eukaryotic protein synthesis and thus are medically important antibiotics. Table 30.10 is a partial list of these inhibitors and their mode of action. The structures of some of these compounds are given in Figure 30.31. As indicated in Table 30.10, many antibiotics inhibit protein synthesis by binding to key rRNA components. The two principal sites for antibiotic binding are the 16S rRNA of the decoding center within the small ribosomal subunit and the PTC of the 23S rRNA in the large subunit. More antibiotics are targeted to the PTC than to the decoding center. Why is rRNA such an optimal target for antibiotics? Generally speaking, it is diffi- cult for organisms to defend themselves against agents that bind rRNA rather than protein. First of all, there are many rRNA genes, so mutation of one gene to antibi- otic resistance (loss in antibiotic binding, for instance) would affect few rRNA mole- cules among the many. Further, four bases comprise RNA (as opposed to 20 amino acids in proteins), so there are fewer possibilities for modification of the rRNA so that the RNA retains its function but loses its ability to bind the antibiotic. The Decoding Site Is a Target of Aminoglycoside Antibiotics Aminoglycoside an- tibiotics are a series of compounds with a 2-deoxy-streptamine (2-DOS) core struc- ture (Figure 30.32a) that is central to their action. These antibiotics bind to an un- paired adenine residue (A 1408 ) within the 16S rRNA decoding site (that part of the 16S rRNA that decodes the mRNA through interactions with the anticodon of the aminoacyl-tRNA in the A site). A 1408 lies within a small internal rRNA loop (Figure 30.32b) that is part of the decoding center. The sugar moiety attached to the 4-position of the 2-DOS core forms H bonds with A 1408 that limit the flexibility of A 1492 and A 1493 on the other side of the small RNA loop; A 1492 and A 1493 interact with codon nucleotides (see Figure 30.21b). This flexibility loss results in errors in trans- lation fidelity, where the code is misread, the wrong amino acid is inserted in the 982 Chapter 30 Protein Synthesis growing polypeptide chain, and the protein being made is nonfunctional. Amino- glycoside antibiotics have been useful probes for decoding center properties. Many Antibiotics Target the PTC and the Peptide Exit Tunnel A chemically di- verse series of antibiotics targets the PTC and/or the adjacent peptide exit tunnel. Among these, the macrolide antibiotics are one of the clinically most important classes; erythromycin (see Figure 30.31), a broadly prescribed macrolide, is repre- sentative of this class. (The newer, semisynthetic ketolide drugs are analogs of the macrolide class and act in a similar manner.) X-ray crystallographic analyses of ribosome–macrolide complexes show the macrolide bound within the peptide exit tunnel, 1 to 1.5 nm below the PTC. Macrolide binding plugs the exit tunnel, pre- venting the movement of the growing peptide chain through the tunnel. Protein synthesis is aborted and the peptidyl-tRNA eventually dissociates from the ribosome. Substituents bound at the 5-position of the macrolide extend outward from the pep- tide exit tunnel toward the PTC. (The 5-position in macrolides is the position where the N(CH 3 ) 2 -containing saccharide ring of erythromycin is attached; see Figure 30.31). All direct interactions of macrolide antibiotics with the ribosome involve 23S rRNA, not ribosomal proteins. Both macrolide and ketolide antibiotics adopt a con- formation within the ribosome that projects a polar face into the peptide exit tun- nel and a nonpolar face toward the tunnel wall. Hydrophobic interactions between the lactone-ring methyl groups and the tunnel wall contribute significantly to the binding energy of these drugs. Residue A 2058 of the prokaryotic 23S rRNA lies at the entrance to the peptide exit tunnel and favors macrolide antibiotic binding. The major large-subunit rRNA of ribosomes found in the cytosol and mitochondria of eukaryotic cells have G at the equivalent position and are unaffected by macrolides. Inhibitor Cells Inhibited Mode of Action Initiation Aurintricarboxylic acid Prokaryotic Prevents IF binding to 30S subunit Kasugamycin Prokaryotic Inhibits fMet-tRNA i f Met binding Streptomycin Prokaryotic Prevents formation of initiation complexes Elongation: Aminoacyl-tRNA Binding Tetracycline Prokaryotic Inhibits aminoacyl-tRNA binding at A site Streptomycin Prokaryotic Codon misreading, insertion of improper amino acid Kirromycin Prokaryotic Binds to EF-Tu, preventing conformational switch from EF-TuϺGTP to EF-TuϺGDP Elongation: Peptide Bond Formation Sparsomycin Prokaryotic Peptidyl transferase inhibitor Chloramphenicol Prokaryotic Binds to 50S subunit, blocks the A site and inhibits peptidyl transferase activity Clindamycin Prokaryotic Binds to 50S subunit, overlapping the A and P sites and blocking peptidyl transferase activity Erythromycin Prokaryotic Blocks the 50S subunit tunnel, causing premature peptidyl-tRNA dissociation Elongation: Translocation Fusidic acid Both Inhibits EF-GϺGDP dissociation from ribosome Thiostrepton Prokaryotic Inhibits ribosome-dependent EF-Tu and EF-G GTPase activity Diphtheria toxin Eukaryotic Inactivates eEF-2 through ADP-ribosylation Cycloheximide Eukaryotic Inhibits translocation of peptidyl-tRNA Premature Termination Puromycin Both Aminoacyl-tRNA analog, binds at A site and acts as peptidyl acceptor, aborting peptide elongation Ribosome Inactivation Ricin Eukaryotic Catalytic inactivation of 28S rRNA via N-glycosidase action on A 4256 TABLE 30.10 Some Protein Synthesis Inhibitors