分子生物学杨洋第七章翻译课件.ppt

Outline Topics 1-4: Four components of translation machinery. T1-mRNA; T2-tRNA; T3-Attachment of amino acids to tRNA (aminoacyl-tRNA synthetases); T4-ribosomeTopic 5-7: Translation process. T5-initiation; T6-elongation; T7-termination. 9/18/20241 Translation processT5: Initiation of translationT6: Elongation of translationT7: termination of translation 9/18/20242Overview of the events of translationTermination Elongation Initiation9/18/20243Topic 5: Initiation of translation9/18/20244Three events must occur:1.ribosome must be recruited to mRNA2. a charged tRNA must be placed into the P site of ribosome3. the ribosome must be precisely positioned over the start codon9/18/20245start codonOne-base shift will result in the synthesis of completely unrelated polypeptide9/18/20246nThe different structure of prokaryotic and eukaryotic mRNA results in distinctly different means of translational initiation9/18/202475-1 prokaryotic mRNAs are recruited to the small subunit by base pairing to rRNAmRNAmRNA/ small subunit complex9/18/202485-2 A specialized tRNA charged with modified methionine binds directly to prokaryotic small subunit Initiator tRNA:fMet-tRNA(甲酰甲硫氨酸甲酰甲硫氨酸)fMet-tRNAfMet9/18/20249modified methionine(甲酰甲硫氨酸甲酰甲硫氨酸)9/18/2024105-3 Three initiation factors (IF) direct the assembly of an initiation complex that contains mRNA and initiator tRNA Translation initiation factors (IF):IF1: prevent tRNA from binding to the A site in the small subunitIF2: a GTPase that interacts with small subunit, IF1 and initiator tRNA (fMet-tRNAfMet); IF2 can facilitate the association of fMet-tRNAfMet with the small subunitIF3: binds to the small subunit and blocks it from reassociating with large subunit, helps to dissociate the 70S ribosome into its large and small subunit9/18/202411Three binding site for tRNAsA site: to bind the aminoacylated-tRNAP-site: to bind the peptidyl-tRNAE-site: to bind the uncharged tRNA 9/18/202412Each of the initiation factors binds at or near one of the three tRNA-binding sites on the small subunit 9/18/202413IF330S small subunit50S large subunitIF1IF2fMet-tRNAfMet50S large subunitIF370S initiation complexIF2-GTPIF2-GDPIF150S large subunitremove9/18/2024145-4 Eukaryotic ribosome are recruited to mRNA by the 5’-CapSimilarity: both use a start codon and initiator tRNA both use initiation factors to form mRNA/small subunit complex before addition of large subunitComparison between prokaryotic and eukaryotic translational initiationDifference: the method of recognizing the mRNA and start codon is fundamentally distinct9/18/202415 Once Kozak sequenceEukaryotic mRNA uses a methylated cap to recruit the ribosome. Once bound, the ribosome scans the mRNA in a 5’-3’ direction to find the AUG start codon---scanning(扫描扫描)Kozak sequence can increase the translation efficiency9/18/20241640S small subunitMet-tRNAMetHairpin: block the binding between mRNA and ribosomeelF4B activates the RNA helicase(RNA 解旋酶解旋酶) in one of the elF4F subunits mRNA/small subunit complexUnwind the hairpin structure9/18/2024175-5 The start codon is found by scanning downstream from 5’-end of mRNA9/18/202418Identification of the start codon bysmall ribosome subunitMet-tRNAMet is placed in the P siteof the 80S initiation complexscanning9/18/202419Scientific question(提出科学问题提出科学问题)nHow the eukaryotic ribosome recognize the start codon AUG?nWhat is the function of initiator tRNA---Met-tRNAMet in the translation initiation of eukaryotic cell?9/18/202420研究论文（原始研究工作）的学习研究论文（原始研究工作）的学习9/18/202421设计实验设计实验实验数据分析实验数据分析得出结论得出结论提出科学问题提出科学问题分析科学问题分析科学问题解决科学问题解决科学问题9/18/202422tRNAiMet functions in directing the scanning ribosome to the start site of translation. Science, Vol 242, Issue 4875, 93-97 nKeywords:nTranslation initiationnRibosomenStart codonnScanning modelnMet-tRNAMet9/18/202423nRibosome binding sites of the prokaryotic type are not present in eukaryotic mRNAnEukaryotic scanning model: nThe 40S ribosomal subunit in association with initiator tRNA (Met-tRNAMet) and initiation factors binds the 5’-cap of the mRNAnmigrates in a 5' to 3' direction, “scanning” for a translational start site9/18/202424AUGEukaryotic translation initiationinitiator tRNA (Met-tRNAMet)40S subunit9/18/202425尚未解决的科学问题：尚未解决的科学问题：nThe precise mechanism by which the scanning ribosome recognizes the first AUG codon has not been discoverednWhat is the role of initiator tRNA (Met-tRNAMet) in the translation initiation process? 9/18/202426How to answer the scientific question(解决科学问题解决科学问题)Establish a good modelDesign the elegant experimentAnalyze the experimental resultsAnswer the scientific question9/18/202427Model: yeast, Saccharomyces cerevisiae (酿酒酵母）酿酒酵母）--model organism of eukaryotes His gene (编码合成组氨酸的基因）编码合成组氨酸的基因）: necessary gene for yeast growth on the basic medium lacking histidine9/18/202428AUG5’His geneWild-type yeast cellCan grow on the basicmedium lacking histidinePhenotype: His+AGG5’His geneMutant yeast cellCan not grow on the basicmedium lacking histidinePhenotype: His-A simple growth test: reflect translation of his gene9/18/202429AGG5’His geneUUG5’His geneAUC5’His geneAAG5’His geneACG5’His geneAUG5’His geneWild-typeMutant-1Mutant-2Mutant-3Mutant-4Mutant-5His+His-His-His-His-His-genotypephenotype9/18/202430UACMetMet-tRNAMet(initiator tRNA )AUG5’His geneWild-type yeast cellGPhenotype: His+9/18/202431UACGMetMet-tRNAMet(initiator tRNA )3’5’mutationUCC3’5’MetmutationtRNA gene was mutatedMutated tRNA9/18/202432UCC3’5’High-copy plasmidOver-expression of mutated tRNA geneAGG5’His geneMutant yeast cell导入导入UUGAUCAAGACGgrowth test on the basic medium lacking histidineMet9/18/202433growthMutated start codon9/18/202434UCC3’5’High-copy plasmidOver-expression of mutated tRNA geneACG5’His geneMutant yeast cell导入导入Can not grow on the basic medium lacking histidinemutated tRNAMet9/18/202435UCC3’5’High-copy plasmidOver-expression of mutated tRNA geneAGG5’His geneMutant yeast cell导入导入Can grow on the basic medium lacking histidinemutated tRNAMet9/18/202436UAC3’5’AGG5’His geneMutant yeast cellCan not grow on the basic medium lacking histidineWild-type tRNAMet9/18/202437AGG5’His geneMutant yeast cell-2AGG+1-28Another experiment:AGG5’His geneMutant yeast cell-1ACG+1-289/18/202438UCC3’5’High-copy plasmidOver-expression of mutated tRNA gene导入导入mutated tRNAAGG5’His geneMutant yeast cell-2AGG+1-28growth test on the basic medium lacking histidineMet9/18/202439UCC3’5’High-copy plasmidOver-expression of mutated tRNA gene导入导入mutated tRNAAGG5’His geneMutant yeast cell-1ACG+1-28growth test on the basic medium lacking histidineMet9/18/202440++++growthHis-His+The upstream AGG out of the reading frame of his geneserves as a site of initiation his genehis geneACG-28The mutant tRNA also directed the ribosome to initiate at an AGG placed in the upstream region of the his gene9/18/202441UCC3’5’High-copy plasmidOver-expression of mutated tRNA gene导入导入mutated tRNAAGG5’His geneMutant yeast cell-2AGG+1-28growth on the basic medium lacking histidine was inhibited Prove the scanning model9/18/202442SD: GUGUGHuman growth hormone geneProduce much human growth hormone protein mutated type cell (16s rRNA: CACAC)9/18/202443nAn anticodon: codon interaction between tRNAMet and the first AUG in the mRNA is important for ribosomal recognition of the translational start site during the scanning process9/18/2024445-6 Translation initiation factors hold eukaryotic mRNAs in circlesIt can explain how the mRNA poly-A tail contributes to the translation efficiency.Fig 14-29elF4F9/18/202445翻译起始的小结翻译起始的小结:nSimilarity and difference between prokaryotic and eukaryotic translational initiationnFunction of translation initiation factors:nIF1,IF2,IF3n eIF1A,eIF3,eIF2, eIF4F, eIF4B9/18/202446Topic 6Elongation of translationThree important events in the elongation of translation9/18/202447进位进位易位易位肽键形成肽键形成9/18/202448Elongation factor:EF-Tu escort aminoacylated-tRNA to the A site of the ribosome 9/18/202449Elongation factor:EF-G stimulates the translocationof tRNA EF-G-GDP9/18/202450肽键形成肽键形成(肽转移酶反应）肽转移酶反应）9/18/202451Ribosome is a ribozyme(核酶核酶):Peptidyl transferase reaction is catalyzed by RNA, specially 23s rRNACatalysis requires distance in the 1-3 Å, and only RNA residues are present 18 Å from the active site.RNA surround the peptidyl transferase center of the large ribosome subunit9/18/202452翻译延伸小结翻译延伸小结:nThree important events in the elongation of translationnFunction of translation elongation factors: EF-Tu, EF-GnPeptidyl transferase reaction, Ribosome is a ribozyme(核酶核酶)9/18/202453Topic 7: termination of translation9/18/202454Overview of the events of translationTermination Elongation Initiation9/18/202455The stop codonUAG, UGA, or UAA (5’-3’)Functions:1.Defines the end of ORF 2.Signal termination of polypeptide synthesis9/18/202456Question:nHow the ribosome can recognize the stop codon?nWhat is the mechanism?9/18/202457UAGpeptidePostulation: Chain-terminating tRNA for recognizing the stop codon ?Release factor (RF, 释放因子）释放因子）is tRNA? Chain-terminatingtRNA?9/18/202458Mario Capecchi’ research work—discovery of release factor (RF, 释放因子）释放因子） model: ribosome and phage R17 mRNA in vitro(体外）体外）translation system9/18/202459The experiment for finding the releasefactor(+RNA酶处理）酶处理）不加苏氨酸不加苏氨酸加加[14C]苏氨酸苏氨酸(+蛋白酶处理）蛋白酶处理）加入核糖体上清液组分加入核糖体上清液组分(Containing RF)（（what is RF?)五肽五肽14C标记的六肽标记的六肽能够释放多肽链能够释放多肽链不能够释放多肽链不能够释放多肽链9/18/202460Discovery:nRelease factor (RF, 释放因子）释放因子）is protein, not RNAnProteins called release factors (RFs) recognize the stop codon and activate the hydrolysis of the polypeptide from peptidyl-tRNA9/18/202461Two classes of release factors nClass I: recognize the stop codon and trigger hydrolysis of the polypeptide from peptidyl-tRNA Prokaryotes: RF1-UAG,UAA RF2-UGA,UAA Eukaryotes: eRF1-UAG,UGA,UAAnClass II: stimulate the dissociation of the class I factors from the ribosome after release of polypeptide Prokaryotes: RF3 Eukaryotes: eRF3 GTP-binding protein 9/18/202462Nirenberg’ research work—more direct prove for the function of RFProkaryotes: RF1-UAG,UAA RF2-UGA,UAA9/18/202463Ribosome-AUG-[3H]fMet-tRNAfMet complex[3H]fMet[3H]fMet9/18/202464[3H]fMet 9/18/202465Further question about Class I release factornHow do release factors recognize the stop codon?nWhat is the mechanism of this recognition?How the ribosome can recognize the stop codon?Proteins called release factors (RFs) recognize the stop codonand activate the hydrolysis of the polypeptide from peptidyl-tRNA9/18/202466nProtein (RF)-RNA (mRNA) interactionnHypothesis: RF mimics the structure and function of tRNARF can compete with tRNA for binding to the ribosome and recognizing codons as tRNA9/18/202467UAGpeptidePostulation: Chain-terminating tRNA for recognizing the stop codon ?Release factor (RF, 释放因子）释放因子）is tRNA? Chain-terminatingtRNA?9/18/202468The site of attachment of amino acidA three-nucleotide-long sequence responsible for recognizing the codon by base pairing with mRNAcloverleaf structure of tRNA9/18/202469nPeptide anticodon(肽反密码子肽反密码子): three-amino acids sequence in RF which can recognize and interact with the stop codon9/18/202470The Tripeptide AnticodonRF1: Pro-Ala-ThrRF2: Ser-Pro-PheCell, 2000, 101: 349-352UAG,UAAUGA,UAA9/18/202471nPeptide anticodon(肽反密码子肽反密码子):nthree-amino acids sequence in RF which can recognize and interact with the stop codonnDetermine the specificity of RF1 and RF2 for recognizing the stop codon 9/18/202472nGGQ sequence (Gly-Gly-Gln): a region of class I release factor which contributes to polypeptide release from tRNAAnother important region in RF:Question: how can we prove that GGQ sequence in RF is really critical for the polypeptide release from tRNA? Genetic method (mutation idea)9/18/202473[3H]fMet Ribosome-AUG-[3H]fMet-tRNAfMet complex[3H]fMet 9/18/202474Class I release factornHow do release factor recognize the stop codon?nProtein-RNA interactionnPeptide anticodon(肽反密码子肽反密码子): three-amino acids sequence which can interact with and recognize the stop codonnGGQ sequence(Gly-Gly-Gln): a region of Class I release factor which contributes to polypeptide release9/18/202475Peptide anticodon9/18/202476Hypothesis about the mechanism of RF: RF mimics the structure and function of tRNA RF can compete with tRNA for binding to the ribosome and recognizing codons as tRNA9/18/202477The Crystal Structure of Human EukaryoticRelease Factor eRF1—Mechanism of Stop CodonRecognition and Peptidyl-tRNA HydrolysisCell, 2000, 100: 311–321 RF mimics the structure and function of tRNA9/18/202478nRF3(eRF3)nGTP-binding proteinnFunction: stimulate the dissociation of the class I release factors from the ribosome after release of polypeptideClass II release factor9/18/202479release of polypeptiderelease of RF1GDPGTP9/18/202480翻译终止小结翻译终止小结:nTwo classes of release factors：：function, mechanismnPeptide anticodon(肽反密码子肽反密码子) and GGQ motif9/18/202481。