分子生物学(杨洋)第七章 翻译-2.ppt
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1、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 t
2、ranslation 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
3、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 init
4、iation,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
5、/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 subu
6、nit, 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 bindin
7、g 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-tRNA
8、fMet,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 subu
9、nit,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
10、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 subuni
11、t 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 quest
12、ion(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 rib
13、osome 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 assoc
14、iation 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
15、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,Ans
16、wer 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
17、 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,ph
18、enotype,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 b
19、asic 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 yea
20、st 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
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