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IF-3 and IF-2 also departfrom the ribosome.A major difference in protein synthesis between prokaryotes andeukaryotes is the existence of at least nine eukaryotic initiation factors. One of these, called cap binding protein or CBPI, binds to the 5'cap of mRNA and facilitates formation of a complex between themRNA and the 40S ribosomal subunit. The mRNA is then scanned toChapter 26 Protein MetabolismTable 26-8 Protein factors required for translation initiation in bacteria and eukaryotesEukaryotesBacteriaFactorFunctionIF-1Stimulates activitiesof IF-2 and IF-3Facilitates binding off Met-tRNA™64 to 30Sribosomal subunitBinds to 30S subunit;prevents prematureassociation of 50SsubunitIF-2IF-3FactorFunctioneIF2*Facilitates binding of initiatingMet-tRNAMet to 40Sribosomal subunitFirst factors to bind 40Ssubunit; facilitate subsequentstepsBinds to 5' cap of mRNABind to mRNA; facilitatescanning of mRNA to locatefirst AUGPromotes dissociation of severalother initiation factors from40S subunit as prelude toassociation of 60S subunit toform 80S initiation complexFacilitates dissociation ofinactive 80S ribosome into40S and 60S subunitseIF3,eIF4CCBPIeIF4A,eIF4B,eIF4FeIF5eIF6^Surprisingly, eIF2 appears to be a multifunctional protein In addition to its role in the initiation of translation, it is also involvedin the splicing of mRNA precursors in the nucleus This finding provides an intriguing link between transcription and translation ineukaryotic cellslocate the first AUG codon, which signals the beginning of the readingframe.
Several additional initiation factors are required in this mRNAscanning reaction, and in assembly of the complete 80S initiation complex in which the initiating Met-tRNAMet and mRNA are bound andready for elongation to proceed. The roles of the various bacterial andeukaryotic initiation factors in the overall process are summarized inTable 26-8. The mechanism by which these proteins act remains avery important area of investigation.In bacteria, the steps in Figure 26-24 result in a functional70S ribosome called the initiation complex, containing the mRNAand the initiating f Met-tRNA™61.
The correct binding of the f MettRNAfMet to the P site in the complete 70S initiation complex is assured by two points of recognition and attachment: the codon-anticodon interaction involving the initiating AUG fixed in the P site, andbinding interactions between the P site and the fMet-tRNAfMetThe initiation complex is now ready for the elongation steps.Peptide Bonds Are Formed during the Elongation StageThe third stage of protein synthesis is elongation, the stepwise addition of amino acids to the polypeptide chain. Again, our discussionfocuses on bacteria. Elongation requires (1) the initiation complex described above, (2) the next aminoacyl-tRNA, specified by the nextcodon in the mRNA, (3) a set of three soluble cytosolic proteins calledelongation factors (EF-Tu, EF-Ts, and EF-G), and (4) GTP.
Threesteps take place in the addition of each amino acid residue, and thiscycle is repeated as many times as there are residues to be added.Part IV Information Pathways920Initiationcomplex(70S)Binding ofnextaminoacyltRNAFigure 26—26 First step in elongation: the bindingof the second aminoacyl-tRNA. The second aminoacyl-tRNA enters bound to EF-Tu (shown as Tu),which also contains bound GTP.
Binding of the second aminoacyl-tRNA to the A site in the ribosomeis accompanied by hydrolysis of the GTP to GDPand Pi, and an EF-Tu*GDP complex leaves the ribosome. The bound GDP is released when the EFTu-GDP complex binds to EF-Ts, and EF-Ts is subsequently released when another molecule of GTPbecomes bound to EF-Tu. This recycles EF-Tu andpermits it to bind another aminoacyl-tRNA.In the first step of the elongation cycle (Fig. 26-26), the next aminoacyl-tRNA is first bound to a complex of EF-Tu containing a molecule of bound GTP.
The resulting aminoacyl-tRNA-EF-Tu»GTP complex is then bound to the A site of the 70S initiation complex. The GTPis hydrolyzed, an EF-Tu»GDP complex is released from the 70S ribosome, and an EF-Tu»GTP complex is regenerated (Fig. 26-26).In the second step, a new peptide bond is formed between theamino acids bound by their tRNAs to the A and P sites on the ribosome(Fig. 26-27). This occurs by the transfer of the initiating Nformylmethionyl group from its tRNA to the amino group of the secondamino acid now in the A site.
The a-amino group of the amino acid inthe A site acts as nucleophile, displacing the tRNA in the P site to formthe peptide bond. This reaction produces a dipeptidyl-tRNA in the Asite and the now "uncharged" (deacylated) tRNA™61 remains bound tothe P site.The enzymatic activity that catalyzes peptide bond formation hashistorically been referred to as peptidyl transferase and was widelyassumed to be intrinsic to one or more of the proteins in the largesubunit. In 1992, Harry Noller and his colleagues discovered that thisactivity was catalyzed not by a protein but by the 23S rRNA, addinganother critical biological function for ribozymes.
As indicated inChapter 25, this startling discovery has important implications for ourunderstanding of the evolution of life on this planet.In the third step of the elongation cycle, called translocation, theribosome moves by the distance of one codon toward the 3' end of themRNA. Because the dipeptidyl-tRNA is still attached to the secondcodon of the mRNA, the movement of the ribosome shifts the dipeptidyl-tRNA from the A site to the P site, and the deacylated tRNA isreleased from the initial P site back into the cytosol. The third codon ofthe mRNA is now in the A site and the second codon in the P site.
Thisshift of the ribosome along the mRNA requires EF-G (also called thetranslocase) and the energy provided by hydrolysis of another moleculeof GTP (Fig. 26-28). A change in the three-dimensional conformationof the entire ribosome is believed to take place at this step in order tomove the ribosome along the mRNA.The ribosome, with its attached dipeptidyl-tRNA and mRNA, isnow ready for another elongation cycle to attach the third amino acidresidue.
This process occurs in precisely the same way as the additionof the second. For each amino acid residue added to the chain, twoGTPs are hydrolyzed to GDP and Pi. The ribosome moves from codon tocodon along the mRNA toward the 3' end, adding one amino acid residue at a time to the growing chain.The polypeptide chain always remains attached to the tRNA of thelast amino acid to have been inserted. This continued attachment to atRNA is the chemical glue that makes the entire process work. Theester linkage between the tRNA and the carboxyl terminus of the polypeptide activates the terminal carboxyl group for nucleophilic attackby the incoming amino acid to form a new peptide bond (as in Fig.26-27).
At the same time, this tRNA represents the only link betweenthe growing polypeptide and the information in the mRNA. As theexisting ester linkage between the polypeptide and tRNA is brokenduring peptide bond formation, a new linkage is formed because eachnew amino acid is itself attached to a tRNA.921Chapter 26 Protein MetabolismIn eukaryotes, the elongation cycle is quite similar.
Three eukaryotic elongation factors called eEFla, eEFl/3y, and eEF2 have functionsanalogous to the bacterial elongation factors EF-Tu, EF-Ts, and EF-G,respectively.PsiteP siteA siteA sitefMet-tRNA™^DipeptidyltRNA2AminoacyltRNA2mRNA 5'TranslocationGTP+ GDP +Peptide bondformation <DipeptidyltRNA25'Direction ofribosome movementFigure 26-27 Second step in elongation: formationof the first peptide bond in bacterial protein synthesis, catalyzed by the 23S rRNA ribozyme. TheiV-formylmethionyl group is transferred to theamino group of the second aminoacyl-tRNA in theA site, forming a dipeptidyl-tRNA.Figure 26-28 Third step in elongation: translocation. The ribosome moves one codon toward the 3'end of mRNA, using energy provided by hydrolysisof GTP bound to EF-G (translocase; shown as G).The dipeptidyl-tRNA is now in the P site, leavingthe A site open for the incoming (third) aminoacyltRNA.922Part IV Information PathwaysProofreading on the Ribosome Is Limited toCodon-Anticodon InteractionsRelease factorbindsThe GTPase activity of EF-Tu makes an important contribution to therate and fidelity of the overall biosynthetic process.
The EF-Tu*GTPcomplex exists for a few milliseconds, and the EF-Tu#GDP complex alsoexists for a similar period before it dissociates. Both of these intervalsprovide an opportunity for the codon-anticodon interactions to be verified (i.e., proofread).
Incorrect aminoacyl-tRNAs normally dissociateduring one of these periods. If the GTP analog GTPyS is used in placeof GTP, hydrolysis is slowed, improving the fidelity but reducing therate of protein synthesis. The process of protein synthesis (includingthe characteristics of codon-anticodon pairing already described) hasclearly been optimized through evolution to balance the requirementsof both speed and fidelity. Improved fidelity might diminish speed,whereas increases in speed would probably compromise fidelity.OGuanosine 5'-O-(3-thiotriphosphate) (GTPrS)Polypeptidyl-tRNAlink hydrolyzedOO0—P—O—P—O—P—O—CH:O"IO"IOHDissociationof componentsOHThis proofreading mechanism establishes only that the propercodon-anticodon pairing has taken place.
The identity of the aminoacids attached to tRNAs is not checked at all on the ribosome. This wasdemonstrated experimentally in 1962 by two research groups led byFritz Lipmann and Seymour Benzer. They isolated enzymaticallyformed Cys-tRNACys and then chemically converted it into AlatRNACys. This hybrid aminoacyl-tRNA, which carries alanine but contains the anticodon for cysteine, was then incubated with a cell-freesystem capable of protein synthesis. The newly synthesized polypeptide was found to contain Ala residues in positions that should havebeen occupied by Cys residues. This important experiment also provided timely proof for Crick's adapter hypothesis.
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