30.4 What Is the Structure of Ribosomes, and How Are They Assembled? 963 rRNAs The rRNAs of E. coli are encoded by a set of seven operons (Figure 30.11). Each of these operons is transcribed into a 30S rRNA precursor that includes several tRNAs. RNase III and other nucleases cleave these precursors to generate 23S, 16S, and 5S rRNA, as well as several tRNAs that are unique to each operon. Transcription of rRNA genes accounts for 80% to 90% of total cellular RNA synthesis. Ribosomal RNAs show extensive potential for intrachain hydrogen bonding and assume sec- ondary structures reminiscent of tRNAs, although substantially more complex (see Figures 11.36 and 11.37). About two-thirds of rRNA is double-helical. Double-helical regions are punctuated by short, single-stranded stretches, generating hairpin con- formations that dominate the molecule; four distinct domains can be discerned in the secondary structure of 16S rRNA and six in 23S rRNA. The three-dimensional struc- tures of both the 30S and 50S ribosomal subunits show that the general shapes of the ribosomal subunits are determined by the conformation of the rRNA molecules within them. Figure 30.12 illustrates the three-dimensional structure of 16S rRNA within the 30S subunit. The overall form of the 30S structure is essentially that of the rRNA (compare Figures 30.12 and 30.13a). The same relationship is true for the 23S plus 5S rRNA and the large ribosomal subunit (compare Figures 11.37 and 30.13b). Ribosomal proteins serve a largely structural role in ribosomes; their primary function is to brace and stabilize the rRNA conformations within the ribosomal subunits. Ribosomes Spontaneously Self-Assemble In Vitro Ribosomal subunit self-assembly is one of the paradigms for the spontaneous forma- tion of supramolecular complexes from their macromolecular components. If the individual proteins and rRNAs composing ribosomal subunits are mixed together in vitro under appropriate conditions of pH and ionic strength, spontaneous self- assembly into functionally competent subunits takes place without the intervention of any additional factors or chaperones. The rRNA acts as a scaffold upon which the various ribosomal proteins convene. Ribosomal proteins bind in a specified order. Bo Sp Sh Be H N P FIGURE 30.12 Tertiary structure of the 16S rRNA within the Thermus thermophilus 30S ribosomal subunit (pdb id ϭ 2J02).This view is of the face that interacts with the 50S subunit (see Figures 30.13 and 30.15).H, head; Be, beak; N, neck; P, platform; Sh, shoulder; Sp, spur; Bo, body. Central protuberance Head Platform Base Decoding center Peptidyl transferase center L7/L12 L1 (a) (b) (c) (d) H 30S 50S Tunnel FIGURE 30.13 Structure of the T. themophilus ribo- somal subunits and 70S ribosome, as deduced by X-ray crystallography. Prominent structural features are labeled. (a) 30S (pdb id ϭ 2J02) and (b) 50S (pdb id ϭ 2J03) subunits.These views show the sides of these two that form the interface between them when they come together to form a 70S subunit (c). (d) is a side view of the 70S ribosome; the white area represents the region where mRNA and tRNAs are bound and peptide bond formation occurs.The tunnel through the 50S subunit that the growing peptide chain transits is shown as a dashed line.The approximate dimensions of the 30S subunit are 5.5 ϫ 22 ϫ 22 nm; the 50S subunit dimensions are 15 ϫ 20 ϫ 20 nm. 964 Chapter 30 Protein Synthesis Ribosomes Have a Characteristic Anatomy Ribosomal subunits have a characteristic three-dimensional architecture that has been revealed by image reconstructions from cryoelectron microscopy, X-ray crys- tallography, and X-ray and neutron solution scattering. Such analyses provide images as depicted in Figure 30.13. The 30S, or small, subunit features a “head” and a “base,” or “body,” from which a “platform” projects. A cleft is defined by the spatial relationship between the head, base, and platform (Figure 30.13a). The mRNA passes across this cleft. The platform represents the central domain of the 30S sub- unit; it contains one-third of the 16S rRNA. This central domain binds mRNA and the anticodon stem-loop end of aminoacyl-tRNAs, providing the framework for de- coding the genetic information in mRNA by mediating codon–anticodon recogni- tion. As such, this central domain of the 30S subunit serves as the decoding center. This center is composed only of 16S rRNA; no ribosomal proteins are involved in de- coding the message. The 50S, or large, subunit is a mitt-like globular structure with three distinctive projections: a “central protuberance,” the “stalk” containing protein L1, and a wing- like ridge known as the “L7/L12 region” (Figure 30.13b). The large subunit binds the aminoacyl-acceptor ends of the tRNAs and is responsible for catalyzing forma- tion of the peptide bond formed between successive amino acids in the polypeptide chain. This catalytic center, the peptidyl transferase, is located at the bottom of a deep cleft. From it, a 10-nm-long tunnel passes outward through the back of the large subunit. The small and large subunits associate with each other in the manner shown in Fig- ure 30.13c and d. The contacts between the 30S and 50S subunits are rather limited, and the subunit interface contains mostly rRNA, with relatively little contribution from ribosomal proteins. The decoding center in the 30S subunit is aligned somewhat with the peptidyl transferase and the tunnel in the large subunit, and the growing peptidyl chain is threaded through this tunnel as protein synthesis proceeds. Even though the ribosomal proteins are arranged peripherally around the rRNAs in ribosomes, rRNA occupies 30% to 40% of the ribosomal subunit surface areas. Both subunits are involved in translocation, the process by which the mRNA moves through the ribosome, one codon at a time. (Although it is physically more likely for the mRNA to move through the ribosome, the descriptions of the events in protein synthesis that follow imply that the ribosome moves along the mRNA.) The Cytosolic Ribosomes of Eukaryotes Are Larger Than Prokaryotic Ribosomes Eukaryotic cells have ribosomes in their mitochondria (and chloroplasts) as well as in the cytosol. The mitochondrial and chloroplastic ribosomes resemble prokary- otic ribosomes in size, overall organization, structure, and function, a fact reflecting the prokaryotic origins of these organelles. Although eukaryotic cytosolic ribosomes are larger and considerably more complex, they retain the “core” structural and functional properties of their prokaryotic counterparts, confirming that the funda- mental ribosome organization and operation has been conserved across evolution- ary time. The rRNA genes of eukaryotes are present in the form of several hundred tandem clusters; these clusters define the nucleolus, a distinct region of the nucleus where these clusters are located and where transcription of rRNA genes occurs. As in prokaryotes, 80% to 90% of eukaryotic transcription is rRNA synthesis. Higher eukaryotes have more complex ribosomes than lower eukaryotes. For example, the yeast cytosolic ribosomes have major rRNAs of 3392 (large subunit) and 1799 nu- cleotides (small subunit); the major rRNAs of mammalian cytosolic ribosomes are 4718 and 1874 nucleotides, respectively. Table 30.6 lists the properties of cytosolic ribosomes in a mammal, the rat. Comparison of base sequences and secondary structures of rRNAs from different organisms suggests that evolution has worked to conserve the secondary structure of these molecules, although not necessarily the nucleotide sequences creating such structure. That is, the retention of a base pair 30.5 What Are the Mechanics of mRNA Translation? 965 at a particular location seems more important than whether the base pair is GϺC or AϺU. 30.5 What Are the Mechanics of mRNA Translation? In translating an mRNA, a ribosome must move along it in the 5Ј→3Ј direction, re- cruiting aminoacyl-tRNAs whose anticodons match up with successive codons and joining amino acids in peptide bonds in a polymerization process that forms a par- ticular protein. Like chemical polymerization processes, protein biosynthesis in all cells is characterized by three distinct phases: initiation, elongation, and termina- tion. At each stage, the energy driving the assembly process is provided by GTP hy- drolysis, and specific soluble protein factors participate in the events. These soluble proteins are often G-protein family members that use the energy released upon hy- drolysis of bound GTP to fuel switchlike conformational changes. Such conforma- tional changes are at the heart of the mechanical steps necessary to move a ribo- some along an mRNA and to deliver an aminoacyl-tRNA into appropriate register with a codon. Initiation involves binding of mRNA by the small ribosomal subunit, followed by association of a particular initiator aminoacyl-tRNA that recognizes the first codon. This codon often lies within the first 30 nucleotides or so of mRNA spanned by the small subunit. The large ribosomal subunit then joins the initiation complex, preparing it for the elongation stage. Elongation includes the synthesis of all peptide bonds from the first to the last. The ribosome remains associated with the mRNA throughout elongation, moving along it and translating its message into an amino acid sequence. This is accom- plished via a repetitive cycle of events in which successive aminoacyl-tRNAs are added to the ribosomeϺmRNA complex as directed by codon binding, the 50S sub- unit catalyzes peptide bond formation, and the polypeptide chain grows by one amino acid at a time. Three tRNA molecules may be associated with the ribosomeϺmRNA complex at any moment. Each lies in a distinct site (Figure 30.14). The A, or acceptor, site is the attachment site for an incoming aminoacyl-tRNA. The P, or peptidyl, site is occupied by peptidyl-tRNA, the tRNA carrying the growing polypeptide chain. The elongation reaction transfers the peptide chain from the peptidyl-tRNA in the P site to the aminoacyl-tRNA in the A site. This transfer occurs through covalent attachment of the ␣-amino group of the aminoacyl-tRNA to the ␣-carboxyl group of the peptidyl- tRNA, forming a new peptide bond. The new, longer peptidyl-tRNA now moves from the A site into the P site as the ribosome moves one codon further along the mRNA. The A site, left vacant by this translocation, can accept the next incoming aminoacyl- tRNA. The E, or exit, site, is transiently occupied by the “unloaded,” or deacylated, tRNA, which has lost its peptidyl chain through the peptidyl transferase reaction. Ribosome Small Subunit Large Subunit Sedimentation coefficient 80S 40S 60S Mass (kD) 4220 1400 2820 Major RNAs 18S ϭ 1874 bases 28S ϭ 4718 bases Minor RNAs 5.8S ϭ 160 bases 5S ϭ 120 bases RNA mass (kD) 2520 700 1820 RNA proportion 60% 50% 65% Protein number 33 polypeptides 49 polypeptides Protein mass (kD) 1700 700 1000 Protein proportion 40% 50% 35% TABLE 30.6 Structural Organization of Mammalian (Rat Liver) Cytosolic Ribosomes (a) 3Ј5Ј AUG UCG GGU Tyr Amino acids UAC AGC Ser Met Ala Lys Peptide bond formation (b) 3Ј5Ј AUG UCG GGUUAC AGC Ser Met Tyr Ala Lys Translocation (ribosome movement) UCG Gly CCA (c) 3Ј5Ј AUG GGUUAC AGC Ser Met Tyr Ala Lys ACTIVE FIGURE 30.14 The basic steps in protein synthesis.The ribosome has three distinct binding sites for tRNA: the A, or acceptor, site; the P, or peptidyl, site; and the E, or exit, site. Test yourself on the concepts in this figure at www.cengage.com/login. 966 Chapter 30 Protein Synthesis These events are summarized in Figure 30.14. The contributions made to each of the three tRNA-binding sites by each ribosomal subunit are shown in Figure 30.15. Termination is triggered when the ribosome reaches a “stop” codon on the mRNA. At this point, the polypeptide chain is released and the ribosomal subunits dissociate from the mRNA. Protein synthesis proceeds rapidly. In vigorously growing bacteria, about 20 amino acid residues are added to a growing polypeptide chain each second. So an average protein molecule of about 300 amino acid residues is synthesized in only 15 seconds. Eukaryotic protein synthesis is only about 10% as fast. Protein syn- thesis is also highly accurate: An inappropriate amino acid is incorporated only once in every 10 4 codons. We focus first on protein synthesis in E. coli, the system for which we know the most. Peptide Chain Initiation in Prokaryotes Requires a G-Protein Family Member The components required for peptide chain initiation include (1) mRNA; (2) 30S and 50S ribosomal subunits; (3) a set of proteins known as initiation factors; (4) GTP; and (5) a specific charged tRNA, f-Met-tRNA i f Met . A discussion of the properties of these components and their interaction follows. Initiator tRNA tRNA i f Met is a particular tRNA for reading an AUG (or GUG, or even UUG) codon that signals the start site, or N-terminus, of a polypeptide chain; the sub- script i signifies “initiation.” This tRNA i f Met does not read internal AUG codons, so it does not participate in chain elongation. Instead, that role is filled by another methionine-specific tRNA, referred to as tRNA Met , which cannot replace tRNA i f Met in peptide chain initiation. (However, both of these tRNAs are loaded with Met by the same methionyl-tRNA synthetase.) The structure of E. coli tRNA i f Met has several distin- guishing features (Figure 30.16). Collectively, these features identify this tRNA as es- sential to initiation and inappropriate for chain elongation. The synthesis of all E. coli polypeptides begins with the incorporation of a modi- fied methionine residue, N-formyl-Met, as N-terminal amino acid. However, in about half of the E. coli proteins, this Met residue is removed once the growing polypep- tide is ten or so residues long; as a consequence, many mature proteins in E. coli lack N-terminal Met. The methionine contributed in peptide chain initiation by tRNA i f Met is unique in that its amino group has been formylated. This reaction is catalyzed by a specific en- zyme, methionyl-tRNA i f Met formyl transferase (Figure 30.17). Note that the addition of the formyl group to the ␣-amino group of Met creates an N-terminal block re- sembling a peptidyl grouping. That is, the initiating Met is transformed into a mini- mal analog of a peptidyl chain. Decoding center Peptidyl transferase center (b) 50S(a) 30S FIGURE 30.15 The three tRNA-binding sites on ribo- somes.The view shows the ribosomal surfaces that form the interface between the 30S (a) and 50S (b) subunits in a 70S ribosome (as if a 70S ribosome has been “opened” like a book to expose facing “pages”).The A (green), P (blue), and E (yellow) sites are occupied by tRNAs.The decoding center on the 30S subunit (b) lies behind the top of the tRNAs in the A and P sites (which is where the anticodon ends of the tRNAs are located). The peptidyl transferase center on the 50S subunit (b) lies at the lower tips (acceptor ends) of the A- and P-site tRNAs. (a: pdb id ϭ 2J02; b: pdb id ϭ 2J03.) E. coli tRNA i fMet A C C A A C G C C C C C G C G G G G p U A A T ψ C A U G C G G A G C C C U C G G G mC A A U A C U A G C C U G G A D 33 G A 30 C 45 50 15 18 10 70 19 40 3G C base pairs 60 57 U N H MetHC O Formylated amino acid No base pairing G C A U G C 7 G m C G G U G C C G C G FIGURE 30.16 The secondary structure of E.coli N-formyl- methionyl-tRNA i fMet .The features distinguishing it from noninitiator tRNAs are highlighted. 30.5 What Are the Mechanics of mRNA Translation? 967 mRNA Recognition and Alignment In order for the mRNA to be translated accu- rately, its sequence of codons must be brought into proper register with the transla- tional apparatus. Recognition of translation initiation sequences on mRNAs involves the 16S rRNA component of the 30S ribosomal subunit. Base pairing between a pyrimidine-rich sequence at the 3Ј-end of 16S rRNA and complementary purine-rich tracts at the 5Ј-end of prokaryotic mRNAs positions the 30S ribosomal subunit in proper alignment with an initiation codon on the mRNA. The purine-rich mRNA se- quence, the ribosome-binding site, is often called the Shine–Dalgarno sequence in honor of its discoverers. Figure 30.18 shows various Shine–Dalgarno sequences found in prokaryotic mRNAs, along with the complementary 3Ј-tract on E. coli 16S rRNA. The 3Ј-end of 16S rRNA resides in the “head” region of the 30S small subunit, and the double helical duplex formed by the Shine-Dalgarno sequence and the 16S rRNA 3Ј-end fits snugly in a chamber between the 30S subunit “head” and “platform” domains. Initiation Factors Initiation involves interaction of the initiation factors (IFs) with GTP, N-formyl-Met-tRNA i f Met , mRNA, and the 30S subunit to give a 30S initiation complex to which the 50S subunit then adds to form a 70S initiation complex. The initiation factors are soluble proteins required for assembly of proper initiation complexes. Their properties are summarized in Table 30.7. + H 3 N S CϳtRNA CH 3 O CH 2 CH 2 C H Met-tRNA i fMet N 10 -formyl-THF THF HC S CϳtRN A CH 3 O CH 2 CH 2 C H N-formyl-Met-tRNA i fMet N O H Note similarity to peptide grouping Met-tRNA i fMet formyl transferase ANIMATED FIGURE 30.17 Methionyl- tRNA i fMet formyl transferase catalyzes the transformyla- tion of methionyl-tRNA i fMet using N 10 -formyl-THF as formyl donor.The tRNA for reading Met codons within a protein (tRNA Met ) is not a substrate for this transformyl- ase. See this figure animated at www.cengage.com/ login. U A C U U A U A C C G U G A U A A C A U A U U C A C A U A A A A A G C U A C C A A C A A G U U C U U C C C A A A A C A A U C C A U A U A A C A G G A G U A G U G A G G G G G A G G G G G G A G G A G G A G G G A G G A G A G G A U A U G U G C G U U G G A G C U C A A A G C G A A U U U A A U A G U C A U G A A U C A A G A A U A U G A G A A A G U U A U C A A C U U C G U G U U C C G U U U C U C A A A A A A G A A A A A A A A U U U U U U U U U U U G G G G G G G G G G G G A A A U G G G G C A C G A C C U C C C A A G A A C U U U A U A A A G C A A C U A U A G U U C U A G C C U C C U U A G G U U A A A A araB galE lacI lacZ Q  phage replicase fX174 phage A protein R17 phage coat protein ribosomal protein S12 ribosomal protein L10 trpE trpL leader 3Ј-end of 16S rRNA 3Ј HO AUUCCUCCACUAG– 5Ј Initiation codon – – – – – – – – – – – – – – – – – – – – – – FIGURE 30.18 Various Shine–Dalgarno sequences rec- ognized by E.coli ribosomes.These sequences lie about ten nucleotides upstream from their respective AUG initiation codon and are complementary to the UCCU core sequence element of E. coli 16S rRNA. GϺU as well as canonical GϺC and AϺU base pairs are involved here. Mass Molecules/ Factor (kD) Ribosome Function IF-1 9 0.15 Binds to 30S A site and prevents tRNA binding IF-2 97 G-protein that binds fMet-tRNA i f Met ; interacts with IF-1 IF-3 23 0.25 Binds to 30S E site; prevents 50S binding TABLE 30.7 Properties of E. coli Initiation Factors 968 Chapter 30 Protein Synthesis Events in Initiation Initiation begins when a 30S subunitϺ(IF-3ϺIF-1) complex binds mRNA and a complex of IF-2, GTP, and f-Met-tRNA i f Met . The sequence of events is summarized in Figure 30.19. Although IF-3 is absolutely essential for mRNA binding by the 30S subunit, it is not involved in locating the proper transla- tion initiation site on the message. The presence of IF-3 on 30S subunits also pre- vents them from reassociating with 50S subunits. IF-3 must dissociate before the 50S subunit will associate with the mRNAϺ30S subunit complex. IF-2 delivers the initiator f-Met-tRNA i f Met in a GTP-dependent process. Apparently, the 30S subunit is aligned with the mRNA such that the initiation codon is situated within the “30S part” of the P site. Upon binding, f-Met-tRNA i f Met enters this 30S portion of the P site. GTP hydrolysis is necessary to form an active 70S ribosome. GTP hydrolysis is triggered when the 50S subunit joins and is accompanied by IF-1 and IF-2 release. The A site of the 70S initiation complex is ready to accept an incoming aminoacyl-tRNA; the 70S ribosome is poised to begin chain elongation. Peptide Chain Elongation Requires Two G-Protein Family Members The requirements for peptide chain elongation are (1) an mRNAϺ70S ribosomeϺ peptidyl-tRNA complex (peptidyl-tRNA in the P site), (2) aminoacyl-tRNAs, (3) a set of proteins known as elongation factors, and (4) GTP. Chain elongation can be divided into three principal steps: 1. Codon-directed binding of the incoming aminoacyl-tRNA at the A site. Decoding center regions of 16S rRNA make sure the proper aminoacyl-tRNA is in the A site by direct surveillance of codon–anticodon base pairing geometry. 2. Peptide bond formation: transfer of the peptidyl chain from the tRNA bearing it to the ONH 2 group of the new amino acid. 3. Translocation of the “one-residue-longer” peptidyl-tRNA to the P site to make room for the next aminoacyl-tRNA at the A site. These shifts are coupled with movement of the ribosome one codon further along the mRNA. The Elongation Cycle The properties of the soluble proteins essential to peptide chain elongation are summarized in Table 30.8. These proteins are present in large quantities, reflecting the great importance of protein synthesis to cell vitality. For example, elongation factor Tu (EF-Tu) is the most abundant protein in E. coli, accounting for 5% of total cellular protein. 5Ј 3Ј 30S initiation complex 70S initiation complex N-formyl– Met A U IF-2 C IF-1 IF-3 30S subunit GTP IF-2 IF-1 IF-3 ++ + P GDP A site formyl-N– Met 5Ј 3Ј Shine–Dalgarno sequence UAG UAG AUC 5Ј 3Ј UAG AUC GTP formyl-N– Met IF-2 IF-1 IF-3 50S subunit ACTIVE FIGURE 30.19 The sequence of events in peptide chain initiation. Test yourself on the concepts in this figure at www.cengage.com/ login. 30.5 What Are the Mechanics of mRNA Translation? 969 Aminoacyl-tRNA Binding EF-Tu binds aminoacyl-tRNA and GTP. There is only one EF-Tu species serving all the different aminoacyl-tRNAs, and aminoacyl-tRNAs are accessible to the A site of active 70S ribosomes only in the form of aminoacyl-tRNAϺEF-TuϺGTP complexes. Once cor- rect base pairing between codon and anticodon has been established within the A site, the GTP is hydrolyzed to GDP and P i by EF-Tu. Because the sites of codon–anticodon recognition and GTP hydrolysis are 7.5 nm apart, conformational changes in the ribosome must convey the information of cognate tRNA recognition to the EF-Tu GTPase site. The aminoacyl end of the tRNA is properly oriented in the peptidyl trans- ferase site of the 50S subunit, and the EF-Tu molecule is released as a EF-TuϺGDP com- plex (Figure 30.20). Elongation factor Ts (EF-Ts) is a guanine-nucleotide exchange factor (GEF) that catalyzes the recycling of EF-Tu by mediating the displacement of GDP and its re- placement by GTP. EF-Ts forms a transient complex with EF-Tu by displacing GDP, whereupon GTP displaces EF-Ts from EF-Tu (Figure 30.20). The Decoding Center: A 16S rRNA Function Analysis of the structures of the 70S ribosomeϺtRNA complexes and isolated 30S subunits has revealed the decoding center in the 30S subunit. This decoding center, where anticodon loops of the A- and P-site tRNAs and the codons of the mRNA are matched up, is primarily a property of 16S rRNA. Figure 30.21 reveals the location of the decoding center and highlights the interface between an mRNA UUU codon, the GAA anticodon of a cognate tRNA Phe , and the elements of the ribosome. 16S rRNA nucleotides A1493, A1492, G530, C518, and C1054 interact extensively with the minor groove of the UUUϺAAG duplex; residues Ser 50 and Pro 48 of ribosomal protein S12 also partici- pate (Figure 30.21b). A1492 and A1493 are part of 16S rRNA helix 44; G530 and C518 are from 16S helix 18. Triggered conformational changes within these 16S rRNA regions are key to codon–anticodon recognition. Peptidyl Transfer Peptidyl transfer, or transpeptidation, is the central reaction of protein synthesis, the actual peptide bond–forming step. No energy input (for ex- ample, in the form of ATP) is needed; the ester bond linking the peptidyl moiety to tRNA is intrinsically reactive. As noted earlier, peptidyl transferase, the activity catalyz- ing peptide bond formation, is associated with the 50S ribosomal subunit. Indeed, this reaction is a property of the 23S rRNA in the 50S subunit. 23S rRNA Is the Peptidyl Transferase Enzyme Peptide bond formation is catalyzed by the large rRNA in the large ribosomal subunit (e.g., the 23S rRNA in prokaryotic 50S ribosomal subunits) through its peptidyl transferase center (PTC). No riboso- mal proteins lie within 1.5 nm of the PTC; thus, none can participate in the reac- tion mechanism. In kinetic terms, the catalytic power of ribosomes is modest: A ri- bosome, through the rRNA comprising its PTC, accelerates the peptide-bond forming reaction rate 4 ϫ 10 6 -fold over the uncatalyzed reaction. Mass Molecules/ Factor (kD) Cell Function EF-Tu 43 70,000 G protein that binds aminoacyl-tRNA and delivers it to the A site EF-Ts 74 10,000 Guanine-nucleotide exchange factor (GEF) that replaces GDP on EF-Tu with GTP EF-G 77 20,000 G protein that promotes translocation of mRNA TABLE 30.8 Properties of E. coli Elongation Factors 970 Chapter 30 Protein Synthesis The Catalytic Power of the Ribosome Comes from Tight Binding of Its Peptidyl- tRNA and Aminoacyl-tRNA Substrates Structurally, the PTC can be described as a funnel-shaped active-site crater where the 3Ј-acceptor ends of the peptidyl-tRNA and aminoacyl-tRNA meet. This crater lies directly above the entrance to the pep- tide exit tunnel. Bases at the 3Ј-ends of the tRNAs base-pair with bases in the PTC. G2251 and G2252 of the PTC (Figure 30.22) base-pair with C75 and C74, respec- tively, of the P-site tRNA. PTC G2553 forms a base pair with C75 of the A-site tRNA. Significantly, PTC C2501 interacts with A76 of the P-site tRNA and nearby U2506 in- teracts with A76 of the A-site tRNA. These interactions and others lead to tight bind- ing of the substrates by the ribosome in a manner such that the reactive groups (aminoacyl and peptidyl) are juxtaposed and properly oriented for reaction to oc- cur. Collectively, functional groups provided by the PTC, the peptidyl-tRNA, and the EF-Tu EF-Tu Aminoacyl-tRNA • Aminoacyl- tRNA P i • EF-Tu • EF-G Process continues Binding Peptidyl transfer Translocation EF-Ts EF-Ts EF-Ts Uncharged tRNA Peptidyl-tRNA Peptidyl-tRNA GTP EF-G • GTP GTP GTP EF-Tu • GDP GTP GDP Peptidyl-tRNA 5Ј 3Ј N-formyl– Met UAG AUCC CGU GGU Ala GA 5Ј 3Ј N-formyl– Met UAG AUCC CGU GGU Ala GA 5Ј 3Ј N-formyl– Met UAG AUC CGU GGU AE 5Ј 3Ј N-formyl– Met CGUUAG GCA AUCϩ GDP ϩ P i GGU Ala Ala GCA ACTIVE FIGURE 30.20 The cycle of events in peptide chain elongation on E.coli ribosomes.The structure at the lower left is an EF-TuϺaminoacyl-tRNA complex (pdb id ϭ 1TTT). Test yourself on the concepts in this figure at www.cengage.com/login. 30.5 What Are the Mechanics of mRNA Translation? 971 aminoacyl-tRNA establish a network of H bonds and electrostatic interactions that stabilize the highly polar transition state (Figure 30.23). In effect, interactions of the tRNAs with the rRNA of the PTC create a highly organized environment that facil- itates peptide bond synthesis. Ribosome-catalyzed protein synthesis relies more on proximity and orientation of substrates than on chemical catalysis. The role of the PTC in protein synthesis is to align the tRNA-linked substrates so that the reaction is facilitated. mRNA 16S RNA A1493 16S RNA G530, C518 C518 16S RNA G530 16S RNA C1054 16S RNA A1492 Codon U1 Codon U2 Codon U3 Ser 50 S12 S12 Pro 48 Anticodon A36 Anticodon A35 Anticodon G34 (a) (b) FIGURE 30.21 The decoding center of the 30S ribo- somal subunit is composed only of 16S rRNA. (a) The 30S subunit, as viewed from the 50S subunit.The circle (cyan) shows the latch structure of the 30S subunit that encircles and encloses the mRNA.The mRNA enters along the path indicated by the arrow and follows a groove along this face of the subunit.The location of the decoding center is indicated by the red circle. (b) 30S ribosomal subunit interactions with the codon– anticodon duplex during cognate tRNA recognition. (Adapted from Figure 2 in Ogle, J. M.,and Ramakrishnan, V., 2005. Structural insights into translational fidelity.Annual Review of Bio- chemistry 74:129–177.) (Adapted from Figure 3 in Schluenzen, F., et al., 2000. Structure of the functionally activated small riboso- mal subunit at 3.3 Å resolution. Cell 102:615–623.) G2252 G2553 G2583 A2450 G2251 C74 C75 C75 P site A site FIGURE 30.22 The peptidyl transferase active site: Base- pairing between cytosine residues of A-site tRNA (yellow) and P-site tRNA (orange) with 23S rRNA bases (pale green) (pdb id ϭ 1VQN).The ␣-amino group of the aminoacyl group on the aminoacyl-tRNA (blue) is posi- tioned for the attack on the ester carbonyl carbon of the peptidyl group of the P-site tRNA (green). (Adapted from Figure 2 in Beringer, M., and Rodnina, M.V., 2007.The ribo- somal peptidyl transferase. Molecular Cell 26:311–321.) 972 Chapter 30 Protein Synthesis Translocation Three things remain to be accomplished in order to return the active 70S ribosomeϺmRNA complex to the starting point in the elongation cycle: 1. The deacylated tRNA must be removed from the P site. 2. The peptidyl-tRNA must be moved (translocated) from the A site to the P site. 3. The ribosome must move one codon down the mRNA so that the next codon is positioned in the A site. The precise events in translocation are still being resolved, but several distinct steps are clear. The acceptor ends (the aminoacylated ends) of both A- and P-site tRNAs in- teract with the PTC of the 50S subunit. Because the growing peptidyl chain doesn’t move during peptidyl transfer, the acceptor end of the A-site aminoacyl-tRNA must move into the P site as its aminoacyl function picks up the peptidyl chain. At the same time, the acceptor end of the deacylated P-site tRNA is shunted into the E site (see Figure 30.15). Then, the mRNA and the anticodon ends of tRNAs move together with respect to the 30S subunit so that the mRNA is passively dragged one codon further through the ribosome. With this movement, the anticodon end of the now one- residue-longer peptidyl-tRNA goes from the A site of the 30S subunit to the P site. Concomitantly, the anticodon end of the deacylated tRNA is moved into the E site. These ratchetlike movements of the 30S subunit relative to the 50S subunit are cat- alyzed by the translocation protein elongation factor G (EF-G), which apparently cou- ples the energy of GTP hydrolysis to movement. Note that translocation of the mRNA relative to the 30S subunit will deliver the next codon to the 30S A site. EF-G binds to the ribosome as an EF-GϺGTP complex. GTP hydrolysis is essential not only for translocation but also for subsequent EF-G dissociation. Because EF-G and EF-Tu compete for a common binding site on the ribosome (the factor-binding center) adjacent to the A site, EF-G release is a prerequisite for return of the 70S ribo- someϺmRNA to the beginning point in the elongation cycle. In this simple model of peptidyl transfer and translocation, the ends of both tRNAs move relative to the two ribosomal subunits in two discrete steps, the ac- ceptor ends moving first and then the anticodon ends. Furthermore, the readjust- ments needed to reposition the ribosomal subunits relative to the mRNA and to one another imply that the 30S and 50S subunits must move relative to one an- other in ratchetlike fashion. This model provides a convincing explanation for why ribo- somes are universally organized into a two-subunit structure: The small and large subunits must move relative to each other, as opposed to moving as a unit, in order to carry out the process of translation. C C A H N H tRNA P O 3Ј 3Ј O peptidyl O C O tRNA A A C C R H C C A N H tRNA P O O – H + peptidyl O C O A C C tRNA A R H peptidyl C C A N H tRNA P OH C H + O O C O tRNA A R H A C C ANIMATED FIGURE 30.23 The protein synthesis reaction proceeds via deprotonation of the ␣-amino group of the aminoacyl-tRNA, followed by nucleophilic attack of the ␣-NH 2 on the peptidyl-tRNA carbonyl carbon to form a highly polar tetrahedral intermediate. Proton transfer to the 3Ј-O of the tRNA that contributed the peptidyl chain leads to release of this tRNA in the deacylated form and formation of a “one- residue-longer”-peptidyl-tRNA. See this figure animated at www.cengage.com/login.