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本课件为《分子生物学》第6章蛋白质合成（Protein synthesis），依据 Gene VII 教材编排，沿The stages、Initiation等小节推进，讲解本章核心概念与分子机制，配逐页图示与文字版。
Chapter 6 Protein synthesis
Figure 6.1 Ribosomes are large ribonucleoprotein particles that contain more RNA than protein and dissociate into large and small subunits.
Figure 6.2 Electron microscopic images of bacterial ribosomes and subunits reveal their shapes. Photographs kindly provided by James Lake.
Figure 6.3 Size comparisons show that the ribosome is large enough to bind tRNAs and mRNA.
Figure 6.4 The ribosome has two sites for binding charged tRNA.
Figure 6.5 Aminoacyl-tRNA enters the A site, receives the polypeptide chain from peptidyl-tRNA, and is transferred into the P site for the enxt cycle of elongation.
Figure 6.6 tRNA and mRNA move through the ribosome in the same direction.
Figure 6.7 Protein synthesis falls into three stages.
Figure 5.9 A ribosome assembles from its subunits on mRNA, translates the nucleotide triplets into protein, and then dissociates from the mRNA.
Initiation factors(IF in prokaryotes, eIF in eukaryotes) are proteins that associate with the small subunit of the ribosome specifically at the stage of initiation of protein synthesis.
Figure 6.8 Initiation requires free ribosome subunits. When ribosomes are released at termination, they dissociate to generate free subunits. Initiation factors are present only on dissociated 30S subunits. When subunits reaassociate to give a functional ribosome at initiation, they release the factors.
Figure 6.15 Ribosome-binding sites on mRNA can be recovered from initiation complexes.
Figure 6.9 Initiation factors stabilize free 30S subunits and bind initiator tRNA to the 30S-mRNA complex.
Figure 6.10 Initiation requires 30S subunits that carry IF-3.
Initiation codon is a special codon (usually AUG) used to start synthesis of a protein.
Figure 6.11 The initiator N-formyl-methionyl-tRNA (f Met-tRNAf) is generated by formyl at ion of methionyl-tRNA, using formyl-tetrahydrofolate as cofact or.
Figure 6.12 Only f Met-tRNAf can be used for initiation by 30S subunits; only other aminoacyl-tRN As (aa-tRNA) can be used for elongation by 70S ribosomes.
Figure 6.13 f Met-tRNAf has unique features that distinguish it as the initiator tRNA.
Figure 6.14 IF-2 is needed to bind f Met-tRNAf to the 30S-mRNA complex. After 50S binding, all IF factors are released and GTP is cleaved.
Figure 6.14 -2.Newly synthesized proteins in bacteria start with formyl-methionine, but the formyl group, and sometimes the methionine, is removed during protein synthesis.
Figure 6.15 Ribosome-binding sites on mRNA can be recovered from initiation complexes.
Figure 6.16 Initiation occurs independently at each cistron in a polycistronic mRNA. When the intercistronic region is longer than the span of the ribosome, dissoci at ion at the termination site is followed by independent reiniti at ion at the next cistron.
Figure 6.19 Several eukaryotic initiation factors are required to unwind mRNA, bind the subunit initiation complex, and support joining with the large subunit.
Figure 6.17 Eukaryotic ribosomes migrate from the 5end of mRNA to the ribosome binding site, which includes an AUG initiation codon.
Figure 6.13 f Met-tRNAf has unique features that distinguish it as the initiator tRNA.
Figure 6.18 In eukaryotic initiation, eIF-2 forms a ternary complex with Met-tRNAf. The ternary complex binds to free 40S subunits, which attach to the 5end of mRNA. Later in the reaction, GTP is hydrolyzed when eIF-2 is released in the form of eIF2-GDP. eIF-2B regenerates the active form.
Figure 6.19 Several eukaryotic initiation factors are required to unwind mRNA, bind the subunit initiation complex, and support joining with the large subunit.
Elongation factors(EF in prokaryotes, eEF in eukaryotes) are proteins that associate with ribosomes cyclically, during addition of each amino acid to the polypeptide chain.
Figure 6.20 EF-Tu-GTP places aminoacyl-tRNA on the ribosome and then is released as EF-Tu-GDP. EF-Ts is required to mediate the replacement of GDP by GTP. The reaction consumes GTP and releases GDP. The only aminoacyl-tRNA that cannot be recognized by EF-Tu-GTP is f Met-tRNAf, whose failure to bind prevents it from responding to internal AUG or GUG codons.
Figure 6.24 Binding of factors EF-Tu and EF-G alternates as ribosomes accept new aminoacyl-tRNA, form peptide bonds, and translocate.
Figure 6.21 Peptide bond formation takes place by reaction between the polypeptide of peptidyl-tRNA in the P site and the amino acid of aminoacyl-tRNA in the A site.
Figure 6.22 Puromyc in mimics aminoacyl-tRNA because it resembles an aromatic amino acid linked to a sugar-base moiety.
Figure 6.23 Models for translocation involve two stages. First, at peptide bond formation the aminoacyl end of the tRNA in the A site becomes located in the P site. Second, the anticodon end of the tRNA becomes located in the P site. Second, the anticodon end of the tRNA becomes located in the P site.
Figure 6.24 Binding of factors EF-Tu and EF-G alternates as ribosomes accept new aminoacyl-tRNA, form peptide bonds, and translocate.
Figure 6.24 The structure of the ternary complex of aminoacyl-tRNA-EF-Tu-GTP (left) resembles the structure of EF-Tu and EF-G are in red and green; the tRNA and the domain resembling it in EF-G are in purple. Photograph kindly provided by Paul Nissen.
Figure 6.25 The structure of the ternary complex of aminoacyl-tRNA.EF-Tu.GTP (left) resembles the structure of EF-G (right). Structurally conserved domains of EF-Tu and EF-G are in red and green; the tRNA and the domain resembling it in EF-G are in purple. Photograph kindly provided by Poul Nissen.
EF-Tu.
GTP (left) resembles the structure of EF-G (right). Structurally conserved domains of EF-Tu and EF-G are in red and green; the tRNA and the domain resembling it in EF-G are in purple. Photograph kindly provided by Poul Nissen.
Figure 6.26 EF-G undergoes a major shift in orientation when translocation occurs.
Missense mutations change a single codon and so may cause the replacement of one amino acid by another in a protein sequence.
Figure 6.27 Molecular mimicry enables the elongation factor Tu-tRNA complex, the translocation factor EF-G, and the release factors RF1/2-RF3 to bind to the same ribosomal site.
Figure 6.28 The RF (release factor) terminates protein synthesis by releasing the protein chain. The RRF (ribosome recycling factor) releases the last tRNA, and EF-G releases RRF, causing the ribosome to dissocuate.
Figure 6.29 The 30S ribosomal subunit is a ribonucleoprotein particle. Proteins are in yellow. Photograph kindly provided by Venkitar a man Ramakrishn an.
Figure 6.30 The 70S ribosome consists of the 50S subunit (blue) and the 30S subunit (purple) with three tRNAs located superficially: yellow in the A site, blue in the P site, and red in the E site. Photograph kindly provided by Harry Noller.
Figure 6.31 The ribosome has several active centers. It may be associated with a membrane. mRNA takes a turn as it passes through the A and P sites, which are angled with regard to each other. The E site lies beyond the P site. The peptidyl transferase site (not shown) stretches across the tops of the A and P sites. Part of the site bound by EF-Tu/G lies at the base of the A and P sites.
Figure 6.32 Some sites in 16S rRNA are protected from chemical probes when 50S subunits join 30S subunits or when aminoacyl-tRNA binds to the A site. Others are the sites of mutations that affect protein synthesis. TERM suppression sites may affect termination at some or several termination codons. The large colored blocks indicate the four domains of the rRNA.
Figure 6.2 Electron microscopic images of bacterial ribosomes and subunits reveal their shapes. Photographs kindly provided by James Lake.
Figure 6.32 Some sites in 16S rRNA are protected from chemical probes when 50S subunits join 30S subunits or when aminoacyl-tRNA binds to the A site. Others are the sites of mutations that affect protein synthesis. TERM suppression sites may affect termination at some or several termination codons. The large colored blocks indicate the four domains of the rRNA.
Figure 6.15 Ribosome-binding sites on mRNA can be recovered from initiation complexes.
Figure 6.33 A change in conform at ion of 16S rRNA may occur during protein synthesis.
Figure 6.34 Codon-anticod on pairing supports interaction with adenines 1492-1493 of 16S rRNA, but mispaired tRNA-mRNA cannot interact.
6.13 23S rRNA has peptidyl transferase activity Figure 6.35 A basic adenine in 23S rRNA could accept a proton from the amino group of the aminoacyl-tRNA. This triggers an attack on the carboxyl group of the peptidyl-tRNA, leading to peptide bond formation.
1.Ribosomes are ribonucleoprotein particles in which a majority of the mass is provided by rRNA. 2.Each subunit contains a single major rRNA, 16S and 23S in prokaryotes, 18S and 28S in eukaryotic cytosol.3.Each subunit has several active centers, concentrated in the translational domain of the ribosome where proteins are synthesized.4.The major rRNAs contain regions that are localized at some of these sites, most notably the mRNA-binding site and P site on the 30S subunit.5.A codon in mRNA is recognized by an aminoacyl-tRNA, which has an anticodon complementary to the codon and carries the amino acid corresponding to the codon.
6.14 Summary6.Ribosomes are released from protein synthesis to enter a pool of free ribosomes that are in equilibrium with separate small and large subunits.7.A ribosome can carry two aminoacyl-tRN As simultaneously: its P site is occupied by a poly peptidyl-tRNA, which carries the polypeptide chain synthesized so far, while the A site is used for entry by an aminoacyl-tRNA carrying the next amino acid to be added to the chain.8.Protein synthesis is an expensive process.9.Additional factors are required at each stage of protein synthesis.10.Prokaryotic EF factors are involved in elongation. EF-Tu binds aminoacyl-tRNA to the 70S ribosome.
Hypothesis Nature416, 281 -28521 March 2002 The trans orientation hypothesis for codon recognition during protein synthesisANNEB.
SIMONSON AND JAMESA.
LAKE Molecular Biology Institute, Human Genetics, and MCD Biology, University of California, Los Angeles, California 90095, USA
The trans orientation model Figure 1 The structures of tRNAs and their orientation on the 30S subunit.
Can the 70S accommodate the ternary complex bound in the D site?Figure 2 The ternary complex docked into the 70S D site.
What is the role of L11 in the trans orientation model? Figure 3 The ternary complex docked in the D site, illustrating steric clash with the 50S L11–RNA complex.
How does the trans orientation hypothesis fit with proofreading?Figure 4 Schematic represent at ion of the ribosome, mRNA and tRNAs during decoding.
How does the trans orientation hypothesis fit with proofreading?http://www.nature.com/nlink/v416/n6878/abs/416281a_fs.html
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