H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 52
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The two subunitscontain rRNAs (red) of different lengths, as well as a different setof proteins. All ribosomes contain two major rRNA molecules18S40S80S(23S and 16S rRNA in bacteria; 28S and 18S rRNA in vertebrates)and a 5S rRNA. The large subunit of vertebrate ribosomes alsocontains a 5.8S rRNA base-paired to the 28S rRNA. The numberof ribonucleotides (rNTs) in each rRNA type is indicated.4.5 • Stepwise Synthesis of Proteins on Ribosomeswith the surface of the rRNAs.
Although the number of protein molecules in ribosomes greatly exceeds the number ofRNA molecules, RNA constitutes about 60 percent of themass of a ribosome.During translation, a ribosome moves along an mRNAchain, interacting with various protein factors and tRNAsand very likely undergoing large conformational changes.Despite the complexity of the ribosome, great progress hasbeen made in determining the overall structure of bacterialribosomes and in identifying various reactive sites. X-raycrystallographic studies on the T.
thermophilus 70S ribosome, for instance, not only have revealed the dimensionsand overall shape of the ribosomal subunits but also have localized the positions of tRNAs bound to the ribosome duringelongation of a growing protein chain. In addition, powerful chemical techniques such as footprinting, which is described in Chapter 11, have been used to identify specificnucleotide sequences in rRNAs that bind to protein or another RNA. Some 40 years after the initial discovery of ribosomes, their overall structure and functioning duringprotein synthesis are finally becoming clear, as we describe inthe next section.KEY CONCEPTS OF SECTION 4.4The Three Roles of RNA in TranslationGenetic information is transcribed from DNA intomRNA in the form of a comma-less, overlapping, degenerate triplet code.■Each amino acid is encoded by one or more threenucleotide sequences (codons) in mRNA. Each codon specifies one amino acid, but most amino acids are encoded bymultiple codons (see Table 4-1).■The AUG codon for methionine is the most common startcodon, specifying the amino acid at the NH2-terminus of aprotein chain.
Three codons (UAA, UAG, UGA) functionas stop codons and specify no amino acids.■A reading frame, the uninterrupted sequence of codonsin mRNA from a specific start codon to a stop codon, istranslated into the linear sequence of amino acids in apolypeptide chain.■Decoding of the nucleotide sequence in mRNA into theamino acid sequence of proteins depends on tRNAs andaminoacyl-tRNA synthetases.■All tRNAs have a similar three-dimensional structurethat includes an acceptor arm for attachment of a specificamino acid and a stem-loop with a three-base anticodonsequence at its ends (see Figure 4-22). The anticodon canbase-pair with its corresponding codon in mRNA.■Because of nonstandard interactions, a tRNA may basepair with more than one mRNA codon; conversely, a particular codon may base-pair with multiple tRNAs.
In each■125case, however, only the proper amino acid is inserted intoa growing polypeptide chain.Each of the 20 aminoacyl-tRNA synthetases recognizesa single amino acid and covalently links it to a cognatetRNA, forming an aminoacyl-tRNA (see Figure 4-21). Thisreaction activates the amino acid, so it can participate inpeptide bond formation.■■ Both prokaryotic and eukaryotic ribosomes—the largeribonucleoprotein complexes on which translation occurs—consist of a small and a large subunit (see Figure 4-24). Eachsubunit contains numerous different proteins and one majorrRNA molecule (small or large).
The large subunit also contains one accessory 5S rRNA in bacteria and two accessoryrRNAs in eukaryotes (5S and 5.8S in vertebrates).Analogous rRNAs from many different species fold intoquite similar three-dimensional structures containing numerous stem-loops and binding sites for proteins, mRNA,and tRNAs. Much smaller ribosomal proteins are associated with the periphery of the rRNAs.■4.5 Stepwise Synthesis of Proteinson RibosomesThe previous sections have introduced the major participantsin protein synthesis—mRNA, aminoacylated tRNAs, and ribosomes containing large and small rRNAs.
We now take adetailed look at how these components are brought togetherto carry out the biochemical events leading to formation ofpolypeptide chains on ribosomes. Similar to transcription,the complex process of translation can be divided into threestages—initiation, elongation, and termination—which weconsider in order. We focus our description on translation ineukaryotic cells, but the mechanism of translation is fundamentally the same in all cells.Methionyl-tRNAiMet Recognizes the AUGStart CodonAs noted earlier, the AUG codon for methionine functionsas the start codon in the vast majority of mRNAs.
A criticalaspect of translation initiation is to begin protein synthesis atthe start codon, thereby establishing the correct readingframe for the entire mRNA. Both prokaryotes and eukaryotes contain two different methionine tRNAs: tRNAiMet caninitiate protein synthesis, and tRNAMet can incorporate methionine only into a growing protein chain.
The sameaminoacyl-tRNA synthetase (MetRS) charges both tRNAswith methionine. But only Met-tRNAiMet (i.e., activated methionine attached to tRNAiMet) can bind at the appropriatesite on the small ribosomal subunit, the P site, to begin synthesis of a polypeptide chain. The regular Met-tRNAMet andall other charged tRNAs bind only to another ribosomal site,the A site, as described later.CHAPTER 4 • Basic Molecular Genetic MechanismseIF6Translation Initiation Usually Occurs Near theFirst AUG Closest to the 5 End of an mRNADuring the first stage of translation, a ribosome assembles,complexed with an mRNA and an activated initiator tRNA,which is correctly positioned at the start codon.
Large andsmall ribosomal subunits not actively engaged in translationare kept apart by binding of two initiation factors, designated eIF3 and eIF6 in eukaryotes. A translation preinitiation complex is formed when the 40S subunit–eIF3 complexis bound by eIF1A and a ternary complex of the MettRNAiMet, eIF2, and GTP (Figure 4-25, step 1). Cells can regulate protein synthesis by phosphorylating a serine residueon the eIF2 bound to GDP; the phosphorylated complex isunable to exchange the bound GDP for GTP and cannotbind Met-tRNAiMet, thus inhibiting protein synthesis.During translation initiation, the 5 cap of an mRNA to betranslated is bound by the eIF4E subunit of the eIF4 capbinding complex. The mRNA-eIF4 complex then associateswith the preinitiation complex through an interaction of theeIF4G subunit and eIF3, forming the initiation complex (Figure 4-25, step 2).
The initiation complex then probably slidesalong, or scans, the associated mRNA as the helicase activityof eIF4A uses energy from ATP hydrolysis to unwindthe RNA secondary structure. Scanning stops when thetRNAiMet anticodon recognizes the start codon, which is thefirst AUG downstream from the 5 end in most eukaryoticmRNAs (step 3). Recognition of the start codon leads to hydrolysis of the GTP associated with eIF2, an irreversible stepthat prevents further scanning.
Selection of the initiating AUGis facilitated by specific surrounding nucleotides called theKozak sequence, for Marilyn Kozak, who defined it: (5)ACCAUGG (3). The A preceding the AUG (underlined) andthe G immediately following it are the most important nucleotides affecting translation initiation efficiency. Once thesmall ribosomal subunit with its bound Met-tRNAiMet is correctly positioned at the start codon, union with the large (60S)ribosomal subunit completes formation of an 80S ribosome.This requires the action of another factor (eIF5) and hydrolysisand J.
D. Richter, 2001, Nature Rev. Mol. Cell Biol. 2:521.]60S40S+60S640S80SeIF3340S340SeIF1A1eIF2•GTP + Met-tRNA iMet(ternary complex)Met 2 -GTP31APreinitiation complexeIF4 (cap-binding complex)+ mRNA2Met 2 -GTP31Am7Gppp4E4A4BAUG(AAA)nInitiation complex2 structureunwinding,scanning, andstart siterecognitionATP3ADP + PiMet FIGURE 4-25 Initiation of translation in eukaryotes.(Inset) When a ribosome dissociates at the termination oftranslation, the 40S and 60S subunits associate with initiationfactors eIF3 and eIF6, forming complexes that can initiateanother round of translation. Steps 1 and 2 : Sequential additionof the indicated components to the 40S subunit–eIF3 complexforms the initiation complex.
Step 3 : Scanning of the mRNA bythe associated initiation complex leads to positioning of thesmall subunit and bound Met-tRNAiMet at the start codon.Step 4 : Association of the large subunit (60S) forms an 80Sribosome ready to translate the mRNA. Two initiation factors, eIF2(step 1 ) and eIF5 (step 4 ) are GTP-binding proteins, whose boundGTP is hydrolyzed during translation initiation. The precise timeat which particular initiation factors are released is not yet wellcharacterized.
See the text for details. [Adapted from R. Mendez60S4G1265eIF1A, eIF3, eIF4 complex,eIF2•GDP + Pi(AAA)n 3AUG60S subunit-eIF6, eIF5•GTP4eIF6, eIF5•GDP + PiMetAUGP80S ribosome(AAA)n4.5 • Stepwise Synthesis of Proteins on RibosomesAt the completion of translation initiation, as noted already, Met-tRNAiMet is bound to the P site on the assembled80S ribosome (Figure 4-26, top). This region of the ribosomeis called the P site because the tRNA chemically linked to thegrowing polypeptide chain is located here.
The secondMeti5EPAEntry of nextaa-tRNA atA siteEF1α •GTP1EGTP hydrolysis,ribosomeconformationalchange▲ FIGURE 4-26 Cycle of peptidyl chain elongationPeptide bondformation▲The correctly positioned eukaryotic 80S ribosome–MettRNAiMet complex is now ready to begin the task of stepwiseaddition of amino acids by the in-frame translation of themRNA. As is the case with initiation, a set of special proteins,termed elongation factors (EFs), are required to carry out thisprocess of chain elongation. The key steps in elongation areentry of each succeeding aminoacyl-tRNA, formation of apeptide bond, and the movement, or translocation, of theribosome one codon at a time along the mRNA.2PA2EF1α •GDP + Pi1 2EPA31EP2AEF2•GTPRibosometranslocation4EF1α •GTPEF1α•GTPDuring Chain Elongation Each IncomingAminoacyl-tRNA Moves ThroughThree Ribosomal Sitesfrom K.
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