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The fact that theamino acids themselves are never checked on the ribosome reinforcesthe central role of aminoacyl-tRNA synthetases in maintaining thefidelity of protein biosynthesis.Termination of Polypeptide Synthesis Requiresa Special SignalFigure 26—29 The termination of protein synthesisin bacteria in response to a termination codon inthe A site. First, a release factor, RFX or RF2 depending on which termination codon is present,binds to the A site. This leads in the second step tohydrolysis of the ester linkage between the nascentpolypeptide and the tRNA in the P site, and releaseof the completed polypeptide. Finally, the mRNA,deacylated tRNA, and release factor leave the ribosome, and the ribosome dissociates into its 30S and50S subunits.Elongation continues until the ribosome adds the last amino acid, completing the polypeptide coded by the mRNA.
Termination, the fourthstage of polypeptide synthesis, is signaled by one of three terminationcodons in the mRNA (UAA, UAG, UGA), immediately following thelast amino acid codon (Box 26-3).In bacteria, once a termination codon occupies the ribosomal A sitethree termination or release factors, the proteins RF1? RF2, andRF 3 , contribute to (1) the hydrolysis of the terminal peptidyl-tRNAbond, (2) release of the free polypeptide and the last tRNA, now uncharged, from the P site, and (3) the dissociation of the 70S ribosomeinto its 30S and 50S subunits, ready to start a new cycle of polypeptidesynthesis (Fig.
26-29). RF1 recognizes the termination codons UAGChapter 26 Protein MetabolismBOX 28-3923Induced Variation in the Genetic Code: Nonsense SuppressionWhen a termination codon is introduced in the interior of a gene by mutation, translation is prematurely halted and the incomplete polypeptidechains are often inactive. Such mutations arecalled nonsense mutations. Restoring the gene toits normal function requires a second mutationthat either converts the termination codon to acodon specifying an amino acid or alternativelysuppresses the effects of the termination codon.The second class of restorative mutations arecalled nonsense suppressors, and they generallyinvolve mutations in tRNA genes that produce altered (suppressor) tRNAs that can recognize thetermination codon and insert an amino acid at thatposition.
Most suppressor tRNAs are created bysingle base substitutions in the anticodons ofminor tRNA species.Suppressor tRNAs constitute an experimentallyinduced variation in the genetic code involving thereading of what are usually termination codons, asis the case for many naturally occurring code variations described in Box 26-2. Nonsense suppression does not completely disrupt informationtransfer in the cell.
This is because there are usually several copies of the genes for some tRNAs inany cell; some of these duplicate genes are weaklyexpressed and account for only a minor part of thecellular pool of a particular tRNA. Suppressormutations usually involve these "minor" tRNAspecies, leaving the major tRNA to read its codonnormally. For example, there are three identicalgenes for tRNA1^" in E. coli, each producing atRNA with the anticodon (5')GUA. One of these isand UAA, and RF 2 recognizes UGA and UAA. Either RFi or RF 2 (asappropriate, depending on which codon is present) binds at a termination codon and induces peptidyl transferase to transfer the growingpeptide chain to a water molecule rather than to another amino acid.The specific function of RF 3 has not been firmly established.
In eukaryotes, a single release factor called eRF recognizes all three terminationcodons.Fidelity in Protein Synthesis Is Energetically ExpensiveThe enzymatic formation of each aminoacyl-tRNA used two highenergy phosphate groups. Additional ATPs are used each time incorrectly activated amino acids are hydrolyzed by the deacylation activityof some aminoacyl-tRNA synthetases (p. 914). One molecule of GTP iscleaved to GDP and Pi during the first elongation step, and anotherGTP is hydrolyzed in the translocation step. Therefore a total of atleast four high-energy bonds is ultimately required for the formation ofeach peptide bond of the completed polypeptide chain.expressed at relatively high levels and thus represents the major tRNA 1 ^ species; the other twogenes are duplicates transcribed in only smallamounts. A change in the anticodon of the tRNAproduct of one of these duplicate tRNA 1 ^ genes,from (5')GUA to (5')CUA, produces a minortRNA1^1" species that will insert tyrosine at UAGstop codons.
This insertion of tyrosine at UAG isinefficient, but can permit production of enoughuseful full-length protein from a gene with a nonsense mutation to allow the cell to live. The majortRNA^ r maintains the normal genetic code for themajority of the proteins.The base change in the tRNA that leads to thecreation of a suppressor tRNA does not alwaysoccur in the anticodon. The suppression of UGAnonsense codons, interestingly, generally involvesthe tRNA^P that normally recognizes UGG.
Thealteration that allows it to read UGA (and insertTrp at these positions) does not occur in the anticodon. Instead, a G ^ A change at position 24 (inan arm of the tRNA somewhat removed from theanticodon) alters the anticodon pairing so that itcan read both UGG and UGA. A similar change isfound in tRNAs involved in the most common naturally occurring variation in the genetic code(UGA = Trp; see Box 26-2).Suppression should lead to many abnormallylong proteins, but, for reasons that are not entirelyclear, this does not always occur. Many details ofthe molecular events that occur during translationtermination and nonsense suppression are notunderstood.Part IV Information Pathways924Incomingribosomal subunits60S140SmRNA-Growingpolypeptide chainDirectionoftranslationThis represents an exceedingly large thermodynamic "push" in thedirection of synthesis: at least 4 x 30.5 = 122 kJ/molofphosphodiesterbond energy is required to generate a peptide bond having a standardfree energy of hydrolysis of only about -21 kJ/mol.
The net free-energychange in peptide-bond synthesis is thus -lOlkJ/mol. Although thislarge energy expenditure may appear wasteful, it is again important toremember that proteins are information-containing polymers. The biochemical problem is not simply the formation of a peptide bond, but theformation of a peptide bond between specific amino acids.
Each of thehigh-energy bonds expended in this process plays a role in a step thatis critical to maintaining proper alignment between each new codon inthe mRNA and the amino acid it encodes at the growing end of thepolypeptide. This energy makes possible the nearly perfect fidelity inthe biological translation of the genetic message of mRNA into theamino acid sequence of proteins.Polysomes Allow Rapid Translation of a Single MessageLarge clusters of 10 to 100 ribosomes can be isolated from either eukaryotic or bacterial cells that are very active in protein synthesis.Such clusters, called polysomes, can be fragmented into individualribosomes by the action of ribonuclease. Furthermore, a connectingfiber between adjacent ribosomes is visible in electron micrographs(Fig. 26-30).
The connecting strand is a single strand of mRNA, beingtranslated simultaneously by many ribosomes, spaced closely together.The simultaneous translation of a single mRNA by many ribosomesallows highly efficient use of the mRNA.In bacteria there is a very tight coupling between transcription andtranslation. Messenger RNAs are synthesized in the 5'—>3' directionand are translated in the same direction. As shown in Figure 26-31,ribosomes begin translating the 5' end of the mRNA before transcription is complete. The situation is somewhat different in eukaryotes,where newly transcribed mRNAs must be transferred out of the nucleus before they can be translated.(a)mRNAPolypeptidechainsRibosomesDirection oftranslation0.25 fimFigure 26-30 A polysome. (a) Four ribosomes areshown translating a eukaryotic mRNA moleculesimultaneously, moving from the 5' end to the 3'end.
(b) Electron micrograph and explanatory diagram of a polysome from the silk gland of a silk-(b)worm larva. The mRNA is being translated bymany ribosomes simultaneously. The polypeptidechains become longer as the ribosomes move toward the 3' end of the mRNA. The final productof this process is silk fibroin.Chapter 26 Protein MetabolismRNA polymeraseDNA duplex5'mRNADirection of transcriptionDirection of translationBacterial mRNAs generally exist for only a few minutes (p. 880)before they are degraded by nucleases. Therefore, in order to maintainhigh rates of protein synthesis, the mRNA for a given protein or set ofproteins must be made continuously and translated with maximumefficiency. The short lifetime of mRNAs in bacteria allows synthesis ofa protein to cease rapidly when it is no longer needed by the cell.Polypeptide Chains Undergo Folding and ProcessingIn the fifth and final step of protein synthesis, the nascent polypeptidechain is folded and processed into its biologically active form.
At somepoint during or after its synthesis, the polypeptide chain spontaneously assumes its native conformation, which permits the maximumnumber of hydrogen bonds and van der Waals, ionic, and hydrophobicinteractions (see Fig. 7-22). In this way, the linear or one-dimensionalgenetic message in the mRNA is converted into the three-dimensionalstructure of the protein. Some newly made proteins do not attain theirfinal biologically active conformation until they have been altered byone or more processing reactions called posttranslational modifications. Both prokaryotic and eukaryotic posttranslational modifications are considered in what follows.Amino-Terminal and Carboxyl-Terminal Modifications Initially,all polypeptides begin with a residue of Af-formylmethionine (in bacteria) or methionine (in eukaryotes). However, the formyl group, theamino-terminal Met residue, and often additional amino-terminal andcarboxyl-terminal residues may be removed enzymatically and thus donot appear in the final functional proteins.In as many as 50% of eukaryotic proteins, the amino group of theamino-terminal residue is acetylated after translation.
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