分子生物学（杨洋）第七章 翻译-2.ppt

2019/6/30,1,Outline,Topics 1-4: Four components of translation machinery. T1-mRNA; T2-tRNA; T3-Attachment of amino acids to tRNA (aminoacyl-tRNA synthetases); T4-ribosome Topic 5-7: Translation process. T5-initiation; T6-elongation; T7-termination.,2019/6/30,2,Translation process T5: Initiation of translation T6: Elongation of translation T7: termination of translation,2019/6/30,3,Overview of the events of translation,Termination,Elongation,Initiation,Topic 5: Initiation of translation,Three events must occur: ribosome must be recruited to mRNA 2. a charged tRNA must be placed into the P site of ribosome 3. the ribosome must be precisely positioned over the start codon,2019/6/30,6,start codon,One-base shift will result in the synthesis of completely unrelated polypeptide,The different structure of prokaryotic and eukaryotic mRNA results in distinctly different means of translational initiation,2019/6/30,8,5-1 prokaryotic mRNAs are recruited to the small subunit by base pairing to rRNA,mRNA,mRNA/ small subunit complex,2019/6/30,9,5-2 A specialized tRNA charged with modified methionine binds directly to prokaryotic small subunit,Initiator tRNA: fMet-tRNA (甲酰甲硫氨酸),fMet-tRNAfMet,2019/6/30,10,modified methionine,(甲酰甲硫氨酸),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 subunit IF2: a GTPase that interacts with small subunit, IF1 and initiator tRNA (fMet-tRNAfMet); IF2 can facilitate the association of fMet-tRNAfMet with the small subunit IF3: binds to the small subunit and blocks it from reassociating with large subunit, helps to dissociate the 70S ribosome into its large and small subunit,2019/6/30,12,Three binding site for tRNAs,A site: to bind the aminoacylated-tRNA P-site: to bind the peptidyl-tRNA E-site: to bind the uncharged tRNA,2019/6/30,13,Each of the initiation factors binds at or near one of the three tRNA-binding sites on the small subunit,IF3,30S small subunit,50S large subunit,IF1,IF2,fMet-tRNAfMet,50S large subunit,IF3,70S initiation complex,IF2-GTP,IF2-GDP,IF1,50S large subunit,remove,5-4 Eukaryotic ribosome are recruited to mRNA by the 5-Cap,Similarity: both use a start codon and initiator tRNA both use initiation factors to form mRNA/small subunit complex before addition of large subunit,Comparison between prokaryotic and eukaryotic translational initiation,Difference: the method of recognizing the mRNA and start codon is fundamentally distinct,2019/6/30,16,Once,Kozak sequence,Eukaryotic 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) Kozak sequence can increase the translation efficiency,2019/6/30,17,40S small subunit,Met-tRNAMet,Hairpin: block the binding between mRNA and ribosome,elF4B activates the RNA helicase (RNA 解旋酶) in one of the elF4F subunits,mRNA/small subunit complex,Unwind the hairpin structure,2019/6/30,18,5-5 The start codon is found by scanning downstream from 5-end of mRNA,2019/6/30,19,Identification of the start codon by small ribosome subunit,Met-tRNAMet is placed in the P site of the 80S initiation complex,scanning,2019/6/30,20,Scientific question(提出科学问题),How the eukaryotic ribosome recognize the start codon AUG? What is the function of initiator tRNA-Met-tRNAMet in the translation initiation of eukaryotic cell?,研究论文（原始研究工作）的学习,2019/6/30,22,设计实验,实验数据分析,得出结论,提出科学问题,分析科学问题,解决科学问题,2019/6/30,23,tRNAiMet functions in directing the scanning ribosome to the start site of translation. Science, Vol 242, Issue 4875, 93-97,Keywords: Translation initiation Ribosome Start codon Scanning model Met-tRNAMet,Ribosome binding sites of the prokaryotic type are not present in eukaryotic mRNA Eukaryotic scanning model: The 40S ribosomal subunit in association with initiator tRNA (Met-tRNAMet) and initiation factors binds the 5-cap of the mRNA migrates in a 5' to 3' direction, “scanning” for a translational start site,AUG,Eukaryotic translation initiation,initiator tRNA (Met-tRNAMet),40S subunit,尚未解决的科学问题：,The precise mechanism by which the scanning ribosome recognizes the first AUG codon has not been discovered What is the role of initiator tRNA (Met-tRNAMet) in the translation initiation process?,2019/6/30,27,How to answer the scientific question(解决科学问题),Establish a good model,Design the elegant experiment,Analyze the experimental results,Answer the scientific question,Model: yeast, Saccharomyces cerevisiae (酿酒酵母） -model organism of eukaryotes,His gene (编码合成组氨酸的基因）: necessary gene for yeast growth on the basic medium lacking histidine,Can grow on the basic medium lacking histidine,Phenotype: His+,AGG,5,His gene,Mutant yeast cell,Can not grow on the basic medium lacking histidine,Phenotype: His-,A simple growth test: reflect translation of his gene,AGG,5,His gene,UUG,5,His gene,AUC,5,His gene,AAG,5,His gene,ACG,5,His gene,AUG,5,His gene,Wild-type,Mutant-1,Mutant-2,Mutant-3,Mutant-4,Mutant-5,His+,His-,His-,His-,His-,His-,genotype,phenotype,UAC,Met,Met-tRNAMet (initiator tRNA ),G,Phenotype: His+,UAC,G,Met,Met-tRNAMet (initiator tRNA ),3,5,mutation,UCC,3,5,Met,mutation,tRNA gene was mutated,Mutated tRNA,High-copy plasmid Over-expression of mutated tRNA gene,AGG,5,His gene,Mutant yeast cell,导入,UUG,AUC,AAG,ACG,growth test on the basic medium lacking histidine,Met,growth,Mutated start codon,High-copy plasmid Over-expression of mutated tRNA gene,ACG,5,His gene,Mutant yeast cell,导入,Can not grow on the basic medium lacking histidine,mutated tRNA,Met,High-copy plasmid Over-expression of mutated tRNA gene,AGG,5,His gene,Mutant yeast cell,导入,Can grow on the basic medium lacking histidine,mutated tRNA,Met,AGG,5,His gene,Mutant yeast cell,Can not grow on the basic medium lacking histidine,Wild-type tRNA,Met,AGG,5,His gene,Mutant yeast cell-2,AGG,+1,-28,Another experiment:,AGG,5,His gene,Mutant yeast cell-1,ACG,+1,-28,High-copy plasmid Over-expression of mutated tRNA gene,导入,mutated tRNA,AGG,5,His gene,Mutant yeast cell-2,AGG,+1,-28,growth test on the basic medium lacking histidine,Met,High-copy plasmid Over-expression of mutated tRNA gene,导入,mutated tRNA,AGG,5,His gene,Mutant yeast cell-1,ACG,+1,-28,growth test on the basic medium lacking histidine,Met,+,+,growth,His-,His+,The upstream AGG out of the reading frame of his gene serves as a site of initiation,his gene,his gene,ACG,-28,The mutant tRNA also directed the ribosome to initiate at an AGG placed in the upstream region of the his gene,High-copy plasmid Over-expression of mutated tRNA gene,导入,mutated tRNA,AGG,5,His gene,Mutant yeast cell-2,AGG,+1,-28,growth on the basic medium lacking histidine was inhibited,Prove the scanning model,SD: GUGUG,Human growth hormone gene,Produce much human growth hormone protein,mutated type E.coli cell (16s rRNA: CACAC),An 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 process,5-6 Translation initiation factors hold eukaryotic mRNAs in circles,It can explain how the mRNA poly-A tail contributes to the translation efficiency.,Fig 14-29,elF4F,2019/6/30,46,翻译起始的小结:,Similarity and difference between prokaryotic and eukaryotic translational initiation Function of translation initiation factors: IF1,IF2,IF3 eIF1A,eIF3,eIF2, eIF4F, eIF4B,Topic 6 Elongation of translation,Three important events in the elongation of translation,进位,易位,肽键形成,Elongation factor: EF-Tu escort aminoacylated -tRNA to the A site of the ribosome,Elongation factor: EF-G stimulates the translocation of tRNA,EF-G-GDP,肽键形成 (肽转移酶反应）,2019/6/30,52,Ribosome is a ribozyme(核酶): Peptidyl transferase reaction is catalyzed by RNA, specially 23s rRNA,Catalysis 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 subunit,2019/6/30,53,翻译延伸小结:,Three important events in the elongation of translation Function of translation elongation factors: EF-Tu, EF-G Peptidyl transferase reaction, Ribosome is a ribozyme(核酶),Topic 7: termination of translation,2019/6/30,55,Overview of the events of translation,Termination,Elongation,Initiation,2019/6/30,56,The stop codon UAG, UGA, or UAA (5-3) Functions: 1.Defines the end of ORF 2.Signal termination of polypeptide synthesis,2019/6/30,57,Question:,How the ribosome can recognize the stop codon? What is the mechanism?,UAG,peptide,Postulation: Chain-terminating tRNA for recognizing the stop codon ? Release factor (RF, 释放因子）is tRNA?,Chain-terminating tRNA,?,Mario Capecchi research workdiscovery of release factor (RF, 释放因子）,model: E.coli ribosome and phage R17 mRNA in vitro(体外）translation system,The experiment for finding the release factor,(+RNA酶处理）,不加苏氨酸,加14C苏氨酸,(+蛋白酶处理）,加入核糖体上清液组分(Containing RF) （what is RF?),五肽,14C标记的六肽,能够释放多肽链,不能够释放多肽链,Discovery:,Release factor (RF, 释放因子）is protein, not RNA Proteins called release factors (RFs) recognize the stop codon and activate the hydrolysis of the polypeptide from peptidyl-tRNA,Two classes of release factors,Class 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,UAA Class II: stimulate the dissociation of the class I factors from the ribosome after release of polypeptide Prokaryotes: RF3 Eukaryotes: eRF3 GTP-binding protein,2019/6/30,63,Nirenberg research workmore direct prove for the function of RF,Prokaryotes: RF1-UAG,UAA RF2-UGA,UAA,Ribosome-AUG-3HfMet-tRNAfMet complex,3HfMet,3HfMet,3HfMet,Further question about Class I release factor,How do release factors recognize the stop codon? What is the mechanism of this recognition?,How the ribosome can recognize the stop codon?,Proteins called release factors (RFs) recognize the stop codon and activate the hydrolysis of the polypeptide from peptidyl-tRNA,Protein (RF)-RNA (mRNA) interaction Hypothesis: RF mimics the structure and function of tRNA RF can compete with tRNA for binding to the ribosome and recognizing codons as tRNA,UAG,peptide,Postulation: Chain-terminating tRNA for recognizing the stop codon ? Release factor (RF, 释放因子）is tRNA?,Chain-terminating tRNA,?,The site of attachment of amino acid,A three-nucleotide-long sequence responsible for recognizing the codon by base pairing with mRNA,cloverleaf structure of tRNA,Peptide anticodon(肽反密码子): three-amino acids sequence in RF which can recognize and interact with the stop codon,Cell, 2000, 101: 349-352,UAG,UAA,UGA,UAA,Peptide anticodon(肽反密码子): three-amino acids sequence in RF which can recognize and interact with the stop codon Determine the specificity of RF1 and RF2 for recognizing the stop codon,GGQ sequence (Gly-Gly-Gln): a region of class I release factor which contributes to polypeptide release from tRNA,Another 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),3HfMet,Ribosome-AUG-3HfMet-tRNAfMet complex,3HfMet,2019/6/30,75,Class I release factor,How do release factor recognize the stop codon? Protein-RNA interaction Peptide anticodon(肽反密码子): three-amino acids sequence which can interact with and recognize the stop codon GGQ sequence(Gly-Gly-Gln): a region of Class I release factor which contributes to polypeptide release,Peptide anticodon,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 tRNA,The Crystal Structure of Human Eukaryotic Release Factor eRF1Mechanism of Stop Codon Recognition and Peptidyl-tRNA Hydrolysis,Cell, 2000, 100: 311321,RF mimics the structure and function of tRNA,2019/6/30,79,RF3(eRF3) GTP-binding protein Function: stimulate the dissociation of the class I release factors from the ribosome after release of polypeptide,Class II release factor,release of polypeptide,release of RF1,GDP,GTP,2019/6/30,81,翻译终止小结:,Two classes of release factors：function, mechanism Peptide anticodon(肽反密码子) and GGQ motif,