分子生物学（杨洋）第七章 翻译课件

1、2020/9/19,1,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.,2020/9/19,2,Translation processT5: Initiation of translationT6: Elongation of translationT7: termination of translation,2020/9/19,3,Overview of the events of translation,Termination,Elongation,Initiation,Topic 5: Initiation of translation,Three events

2、 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,2020/9/19,6,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,2020/9/19,8,5-1 prokaryotic mRNAs are recruited to the small subunit by base pairing to rRNA,mRN

3、A,mRNA/ small subunit complex,2020/9/19,9,5-2 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,2020/9/19,10,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

4、, 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,2020/9/19,12,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,2020/9/19,13,Each of the initiation factors binds at or near one of the three

5、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 re

6、cognizing the mRNA and start codon is fundamentally distinct,2020/9/19,16,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,2020/9/19,17,40S 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 c

7、omplex,Unwind the hairpin structure,2020/9/19,18,5-5 The start codon is found by scanning downstream from 5-end of mRNA,2020/9/19,19,Identification of the start codon by small ribosome subunit,Met-tRNAMet is placed in the P site of the 80S initiation complex,scanning,2020/9/19,20,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,2020/9/19,22,设计

8、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,2020/9/19,23,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

9、 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?,2020/9/19,27,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 o

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