H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 53
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H. Nierhaus et al., 2000, in R. A. Garrett et al., eds., TheRibosome: Structure, Function, Antibiotics, and Cellular Interactions,ASM Press, p. 319.]EF1α •GTP21during translation in eukaryotes. Once the 80S ribosomewith Met-tRNAiMet in the ribosome P site is assembled (top), aternary complex bearing the second amino acid (aa2) codedby the mRNA binds to the A site (step 1 ). Following aconformational change in the ribosome induced by hydrolysis ofGTP in EF1GTP (step 2 ), the large rRNA catalyzes peptidebond formation between Meti and aa2 (step 3 ). Hydrolysis ofGTP in EF2GTP causes another conformational change in theribosome that results in its translocation one codon along themRNA and shifts the unacylated tRNAiMet to the E site and thetRNA with the bound peptide to the P site (step 4 ).
The cyclecan begin again with binding of a ternary complex bearing aa3to the now-open A site. In the second and subsequentelongation cycles, the tRNA at the E site is ejected duringstep 2 as a result of the conformational change induced byhydrolysis of GTP in EF1GTP. See the text for details. [Adapted380S ribosome4EF2•GDP + Pi12EPAMEDIA CONNECTIONS1Focus Animation: Protein Synthesisof a GTP associated with it (step 4). Coupling the joiningreaction to GTP hydrolysis makes this an irreversible step, sothat the ribosomal subunits do not dissociate until the entiremRNA is translated and protein synthesis is terminated. As discussed later, during chain elongation, the growing polypeptideremains attached to the tRNA at this P site in the ribosome.The eukaryotic protein-synthesizing machinery beginstranslation of most cellular mRNAs within about 100 nucleotides of the 5 capped end as just described.
However,some cellular mRNAs contain an internal ribosome entry site(IRES) located far downstream of the 5 end. In addition,translation of some viral mRNAs, which lack a 5 cap, is initiated at IRESs by the host-cell machinery of infected eukaryotic cells. Some of the same translation initiation factorsthat assist in ribosome scanning from a 5 cap are requiredfor locating an internal AUG start codon, but exactly how anIRES is recognized is less clear. Recent results indicate thatsome IRESs fold into an RNA structure that binds to a thirdsite on the ribosome, the E site, thereby positioning a nearbyinternal AUG start codon in the P site.127128CHAPTER 4 • Basic Molecular Genetic Mechanismsaminoacyl-tRNA is brought into the ribosome as a ternarycomplex in association with EF1GTP and becomes boundto the A site, so named because it is where aminoacylatedtRNAs bind (step 1).
If the anticodon of the incoming (second) aminoacyl-tRNA correctly base-pairs with the secondcodon of the mRNA, the GTP in the associated EF1GTP ishydrolyzed. The hydrolysis of GTP promotes a conformational change in the ribosome that leads to tight binding ofthe aminoacyl-tRNA in the A site and release of the resultingEF1GDP complex (step 2 ). This conformational changealso positions the aminoacylated 3 end of the tRNA in the Asite in close proximity to the 3 end of the Met-tRNAiMet inthe P site.
GTP hydrolysis, and hence tight binding, does notoccur if the anticodon of the incoming aminoacyl-tRNA cannot base-pair with the codon at the A site. In this case, theternary complex diffuses away, leaving an empty A site thatcan associate with other aminoacyltRNA–EF1GTP complexes until a correctly base-paired tRNA is bound. This phenomenon contributes to the fidelity with which the correctaminoacyl-tRNA is loaded into the A site.With the initiating Met-tRNAiMet at the P site and thesecond aminoacyl-tRNA tightly bound at the A site, the amino group of the second amino acid reacts with the “activated” (ester-linked) methionine on the initiator tRNA,forming a peptide bond (Figure 4-26, step 3 ; see Figures4-19 and 4-21). This peptidyltransferase reaction is catalyzedby the large rRNA, which precisely orients the interactingatoms, permitting the reaction to proceed. The catalytic ability of the large rRNA in bacteria has been demonstrated bycarefully removing the vast majority of the protein fromlarge ribosomal subunits.
The nearly pure bacterial 23SrRNA can catalyze a peptidyltransferase reaction betweenanalogs of aminoacylated-tRNA and peptidyl-tRNA. Furthersupport for the catalytic role of large rRNA in protein synthesis comes from crystallographic studies showing that noproteins lie near the site of peptide bond synthesis in the crystal structure of the bacterial large subunit.Following peptide bond synthesis, the ribosome istranslocated along the mRNA a distance equal to onecodon. This translocation step is promoted by hydrolysis of the GTP in eukaryotic EF2GTP.
As a result oftranslocation, tRNAiMet, now without its activated methionine, is moved to the E (exit) site on the ribosome;concurrently, the second tRNA, now covalently bound toa dipeptide (a peptidyl-tRNA), is moved to the P site(Figure 4-26, step 4). Translocation thus returns the ribosome conformation to a state in which the A site isopen and able to accept another aminoacylated tRNAcomplexed with EF1GTP, beginning another cycle ofchain elongation.Repetition of the elongation cycle depicted in Figure 4-26adds amino acids one at a time to the C-terminus of thegrowing polypeptide as directed by the mRNA sequenceuntil a stop codon is encountered. In subsequent cycles,the conformational change that occurs in step 2 ejects theunacylated tRNA from the E site.
As the nascent polypeptidechain becomes longer, it threads through a channel in thelarge ribosomal subunit, exiting at a position opposite theside that interacts with the small subunit (Figure 4-27).The locations of tRNAs bound at the A, P, and E sites arevisible in the recently determined crystal structure of the bacterial ribosome (Figure 4-28). Base pairing is also apparentbetween the tRNAs in the A and P sites with their respectivecodons in mRNA (see Figure 4-28, inset). An RNA-RNAhybrid of only three base pairs is not stable under physio-(a)50S30S70S(b)Polypeptide50SEPA30S5mRNA3▲ FIGURE 4-27 Low-resolution model of E.
coli 70S ribosome. (a) Top panels show cryoelectron microscopic images ofE. coli 70S ribosomes and 50S and 30S subunits. Bottom panelsshow computer-derived averages of many dozens of images inthe same orientation. (b) Model of a 70S ribosome based on thecomputer-derived images and on chemical cross-linking studies.Three tRNAs are superimposed on the A (pink), P (green), and E(yellow) sites. The nascent polypeptide chain is buried in a tunnelin the large ribosomal subunit that begins close to the acceptorstem of the tRNA in the P site. [See I. S. Gabashvili et al., 2000, Cell100:537; courtesy of J. Frank.]4.5 • Stepwise Synthesis of Proteins on RibosomesEP129A(a) 70S▲ FIGURE 4-28 Structure of T.
thermophilus 70S ribosomeas determined by x-ray crystallography. (a) Model of the entireribosome viewed from the side diagrammed in Figure 4-26with large subunit on top and small subunit below. The tRNAspositioned at the A (blue), P (yellow), and E (green) sites arevisible in the interface between the subunits with their anticodonloops pointing down into the small subunit. 16S rRNA is cyan;23S rRNA, purple; 5S rRNA, pink; mRNA, red; small ribosomalproteins, dark gray; and large ribosomal proteins, light gray.
Notethat the ribosomal proteins are located primarily on the surfaceof the ribosome and the rRNAs on the inside. (b) View of thelarge subunit rotated 90° about the horizontal from the view in(a) showing the face that interacts with the small subunit. ThetRNA anticodon loops point out of the page. In the intactlogical conditions.
However, multiple interactions betweenthe large and small rRNAs and general domains of tRNAs(e.g., the D and TCG loops) stabilize the tRNAs in the Aand P sites, while other RNA-RNA interactions sense correctcodon-anticodon base pairing, assuring that the genetic codeis read properly.Translation Is Terminated by Release FactorsWhen a Stop Codon Is ReachedThe final stage of translation, like initiation and elongation,requires highly specific molecular signals that decide the fateof the mRNA–ribosome–tRNA-peptidyl complex.
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