H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 54
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Two typesof specific protein release factors (RFs) have been discovered.Eukaryotic eRF1, whose shape is similar to that of tRNAs,apparently acts by binding to the ribosomal A site and recognizing stop codons directly. Like some of the initiation andelongation factors discussed previously, the second eukaryotic release factor, eRF3, is a GTP-binding protein. TheeRF3GTP acts in concert with eRF1 to promote cleavage ofthe peptidyl-tRNA, thus releasing the completed protein(b) 50S(c) 30Sribosome, these extend into the small subunit where theanticodons of the tRNAs in the A and P sites base-pair withcodons in the mRNA. (c) View of the face of the small subunitthat interacts with the large subunit in (b).
Here the tRNAanticodon loops point into the page. The TCG loops andacceptor stems extend out of the page and the 3 CCA ends ofthe tRNAs in the A and P sites point downward. Note the closeopposition of the acceptor stems of tRNAs in the A and P sites,which allows the amino group of the acylated tRNA in the A siteto react with the carboxyl-terminal C of the peptidyl-tRNA in theP site (see Figure 4-19). In the intact ribosome, these are locatedat the peptidyltransferase active site of the large subunit.
[Adaptedfrom M. M. Yusupov et al., 2001, Science 292:883.]chain (Figure 4-29). Bacteria have two release factors (RF1and RF2) that are functionally analogous to eRF1 and aGTP-binding factor (RF3) that is analogous to eRF3.After its release from the ribosome, a newly synthesizedprotein folds into its native three-dimensional conformation,a process facilitated by other proteins called chaperones(Chapter 3).
Additional release factors then promote dissociation of the ribosome, freeing the subunits, mRNA, and terminal tRNA for another round of translation.We can now see that one or more GTP-binding proteinsparticipate in each stage of translation. These proteins belong to the GTPase superfamily of switch proteins that cyclebetween a GTP-bound active form and GDP-bound inactiveform (see Figure 3-29). Hydrolysis of the bound GTP isthought to cause conformational changes in the GTPase itselfor other associated proteins that are critical to various complex molecular processes. In translation initiation, for instance, hydrolysis of eIF2GTP to eIF2GDP prevents furtherscanning of the mRNA once the start site is encountered andallows binding of the large ribosomal subunit to the smallsubunit (see Figure 4-25, step 3 ).
Similarly, hydrolysis of130CHAPTER 4 • Basic Molecular Genetic Mechanisms53UAAEPAeRF1 + eRF3•GTPeRF3 -GTPeRF15UAAEPeptidyl-tRNAcleavageP3AeRF1 + eRF3•GDP + Pidisengage from the 3 end of an mRNA. Simultaneous translation of an mRNA by multiple ribosomes is readily observable in electron micrographs and by sedimentation analysis,revealing mRNA attached to multiple ribosomes bearingnascent growing polypeptide chains.
These structures, referred to as polyribosomes or polysomes, were seen to be circular in electron micrographs of some tissues. Subsequentstudies with yeast cells explained the circular shape of polyribosomes and suggested the mode by which ribosomes recycle efficiently.These studies revealed that multiple copies of a cytosolicprotein found in all eukaryotic cells, poly(A)-binding protein(PABPI), can interact with both an mRNA poly(A) tail andthe 4G subunit of yeast eIF4. Moreover, the 4E subunit ofyeast eIF4 binds to the 5 end of an mRNA. As a result ofthese interactions, the two ends of an mRNA molecule canbe bridged by the intervening proteins, forming a “circular”mRNA (Figure 4-30).
Because the two ends of a polysomeare relatively close together, ribosomal subunits that disengage from the 3 end are positioned near the 5 end, facilitating re-initiation by the interaction of the 40S subunit witheIF4 bound to the 5 cap. The circular pathway depicted inFigure 4-31, which may operate in many eukaryotic cells,would enhance ribosome recycling and thus increase the efficiency of protein synthesis.▲ FIGURE 4-29 Termination of translation in eukaryotes.When a ribosome bearing a nascent protein chain reaches astop codon (UAA, UGA, UAG), release factor eRF1 enters theribosomal complex, probably at or near the A site together witheRF3GTP. Hydrolysis of the bound GTP is accompanied bycleavage of the peptide chain from the tRNA in the P site andrelease of the tRNAs and the two ribosomal subunits.EF2GTP to EF2GDP during chain elongation leads totranslocation of the ribosome along the mRNA (see Figure4-26, step 4).Polysomes and Rapid Ribosome RecyclingIncrease the Efficiency of TranslationAs noted earlier, translation of a single eukaryotic mRNAmolecule to yield a typical-sized protein takes 30–60 seconds.
Two phenomena significantly increase the overall rateat which cells can synthesize a protein: the simultaneoustranslation of a single mRNA molecule by multiple ribosomes and rapid recycling of ribosomal subunits after they▲ EXPERIMENTAL FIGURE 4-30 Eukaryotic mRNA formsa circular structure owing to interactions of three proteins.In the presence of purified poly(A)-binding protein I (PABPI),eIF4E, and eIF4G, eukaryotic mRNAs form circular structures,visible in this force-field electron micrograph. In these structures,protein-protein and protein-mRNA interactions form a bridgebetween the 5 and 3 ends of the mRNA as diagrammed inFigure 4-31.
[Courtesy of A. Sachs.]4.6 • DNA Replication60SPABPI PABPImRNAAAA A AA3 A A80SKEY CONCEPTS OF SECTION 4.5Stepwise Synthesis of Proteins on RibosomesOf the two methionine tRNAs found in all cells, onlyone (tRNAiMet) functions in initiation of translation.▲ FIGURE 4-31 Model of protein synthesis oncircular polysomes and recycling of ribosomalsubunits. Multiple individual ribosomes cansimultaneously translate a eukaryotic mRNA, shownhere in circular form stabilized by interactions betweenproteins bound at the 3 and 5 ends.
When aribosome completes translation and dissociates fromthe 3 end, the separated subunits can rapidly find thenearby 5 cap (m7G) and initiate another round ofsynthesis.▲elF4Gbring the two ends of a polyribosome close together,thereby promoting the rapid recycling of ribosomal subunits, which further increases the efficiency of protein synthesis (see Figure 4-31).■Each stage of translation—initiation, chain elongation,and termination—requires specific protein factors including GTP-binding proteins that hydrolyze their bound GTPto GDP when a step has been completed successfully.■During initiation, the ribosomal subunits assemble nearthe translation start site in an mRNA molecule with thetRNA carrying the amino-terminal methionine (Met-tRNAiMet)base-paired with the start codon (Figure 4-25).■Chain elongation entails a repetitive four-step cycle: loosebinding of an incoming aminoacyl-tRNA to the A site onthe ribosome; tight binding of the correct aminoacyl-tRNAto the A site accompanied by release of the previously usedtRNA from the E site; transfer of the growing peptidyl chainto the incoming amino acid catalyzed by large rRNA; andtranslocation of the ribosome to the next codon, therebymoving the peptidyl-tRNA in the A site to the P site andthe now unacylated tRNA in the P site to the E site (seeFigure 4-26).■In each cycle of chain elongation, the ribosome undergoestwo conformational changes monitored by GTP-bindingproteins.
The first permits tight binding of the incomingaminoacyl-tRNA to the A site and ejection of a tRNA fromthe E site, and the second leads to translocation.■Termination of translation is carried out by two typesof termination factors: those that recognize stop codonsand those that promote hydrolysis of peptidyl-tRNA (seeFigure 4-29).■The efficiency of protein synthesis is increased by the simultaneous translation of a single mRNA by multiple ribosomes. In eukaryotic cells, protein-mediated interactions■4.6 DNA ReplicationNow that we have seen how genetic information encoded inthe nucleotide sequences of DNA is translated into the structures of proteins that perform most cell functions, we can appreciate the necessity of the precise copying of DNAsequences during DNA replication (see Figure 4-1, step 4).The regular pairing of bases in the double-helical DNA structure suggested to Watson and Crick that new DNA strandsare synthesized by using the existing (parental) strands astemplates in the formation of new, daughter strands complementary to the parental strands.This base-pairing template model theoretically could proceed either by a conservative or a semiconservative mechanism.
In a conservative mechanism, the two daughter strandswould form a new double-stranded (duplex) DNA moleculeand the parental duplex would remain intact. In a semiconservative mechanism, the parental strands are permanentlyseparated and each forms a duplex molecule with the daughter strand base-paired to it. Definitive evidence that duplexDNA is replicated by a semiconservative mechanism camefrom a now classic experiment conducted by M. Meselsonand W. F.
Stahl, outlined in Figure 4-32.Copying of a DNA template strand into a complementary strand thus is a common feature of DNA replication andtranscription of DNA into RNA. In both cases, the information in the template is preserved. In some viruses, singlestranded RNA molecules function as templates for synthesisof complementary RNA or DNA strands. However, the vastpreponderance of RNA and DNA in cells is synthesized frompreexisting duplex DNA.MEDIA CONNECTIONS5m7G elF4EOverview Animation: Life Cycleof an mRNA40S131132CHAPTER 4 • Basic Molecular Genetic Mechanisms(b) Actual results(a) Predicted resultsConservative mechanismDensitySemiconservative mechanismDensityGeneration0Parental strandssynthesized in 15N0.3HHH0.7H1.0NewNewOld1.1After firstdoubling in 14N1.51.9HHLLHLLH2.53.04.1After seconddoubling in 14N0 and 1.9mixed0 and 4.1mixedHHLLLLLLLL▲ EXPERIMENTAL FIGURE 4-32 The Meselson-Stahlexperiment showed that DNA replicates by asemiconservative mechanism. In this experiment, E.
colicells initially were grown in a medium containing ammoniumsalts prepared with “heavy” nitrogen (15N) until all the cellularDNA was labeled. After the cells were transferred to a mediumcontaining the normal “light” isotope (14N), samples wereremoved periodically from the cultures and the DNA in eachsample was analyzed by equilibrium density-gradientcentrifugation (see Figure 5-37). This technique can separateheavy-heavy (H-H), light-light (L-L), and heavy-light (H-L) duplexesinto distinct bands. (a) Expected composition of daughter duplexmolecules synthesized from 15N-labeled DNA after E.
coli cellsare shifted to 14N-containing medium if DNA replication occursby a conservative or semiconservative mechanism. Parentalheavy (H) strands are in red; light (L) strands synthesized aftershift to 14N-containing medium are in blue. Note that theconservative mechanism never generates H-L DNA and thatthe semiconservative mechanism never generates H-H DNAbut does generate H-L DNA during the second and subsequentdoublings. With additional replication cycles, the 15N-labeled (H)strands from the original DNA are diluted, so that the vast bulkof the DNA would consist of L-L duplexes with eitherDNA Polymerases Require a Primerto Initiate ReplicationAnalogous to RNA, DNA is synthesized from deoxynucleoside 5-triphosphate precursors (dNTPs). Also like RNA synthesis, DNA synthesis always proceeds in the 5n3HLLHLLL-L H-L H-HL-L H-LH-Hmechanism.
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