Chapter 6(1).pptx

Chapter 6Ribosome，synthesis and transport of proteins,Ribosome:structure and function Transport of proteinsFate of proteins,OUTLINE,1.Ribosome(核糖体),The ribosome is a small,campact ribonucleoprotein particle(核糖核蛋白颗粒)composed of RNAs and proteins，and involved in protein synthesis.Ribosome for short,Prokaryotes,1)Ribosome structure,(1)Shape of ribosome,-no membrane structure-two subunits,(2)Chemical component of ribosomes,Each subunit consists of rRNAs and proteins RNAs inside the subunit,proteins over the surface(The proteins of the small subunit is named as S1,S2,.and the proteins of the large subunit is named as L1,L2,.).,(31),Main types of ribosomes,3.9103-4.5103kDa,rRNAs?,16S rRNA：1457-1544 nt，the conserved sequence of ACCUCCUUA at the 3end is complementary with the purine-rich SD sequence.,18S rRNA:the homologous nucleotide sequence to 16S rRNA at the 3end.,Sequence characteristics of rRNAs,5S rRNA(细菌)：120 or 116 nt,two highly conserved sequences,one CGAAC sequence may recognise tRNA,the other may interact with GTCG sequence of TC loop.,5.8S rRNA：160nt，the CGAAC sequence similar to highly conserved one of 5S rRNA.23S rRNA：2904 nt,the sequence complementary with tRNAfMet sequence.28S rRNA：3890-4700nt，unknown function.,TC臂负责和核糖体上的rRNA 识别结合,Compositions of ribosome from different sources,SourceRibosomeSubunits ribosomal RNAs Cytoplasm 80S 60S(大亚基)28S(Eukaryote)40S(小亚基)18S,5.8S,5S Cytoplasm 70S 50S(大亚基)23S(Prokaryote)30S(小亚基)16S,5S Mitochondrion 55-60S 45S(大亚基)16S(Mamalian)35S(小亚基)12S Mitochondrion 75S 53S(大亚基)21S(Yeast)35S(小亚基)14S Mitochondrion 78S 60S(大亚基)26S(Plant)45S(小亚基)18S,5SChloroplast 70S 50S(大亚基)23S 30S(小亚基)16S,5S,2)Biogenesis of Ribosome,Synthesis of ribosomal proteins and rRNAs Assembly of subunits,(1)Synthesis and processing of rRNAs Eukaryotic cells:rRNA genes of 18S,5.8S and 28S rRNA,as a transcription unit,are tandemly(串联地)repeated 1001,000 times on chromosomes.Transcription RNA polymerase I primary transcript:34S45S,13,pre-rRNA in different species,Species pre-rRNA Drosophila(果蝇)34S S.pombe(裂殖酵母)37S Tobacco 38S Frog 40S Chicken 45S Mouse 45S Human 45S,14,Processing of 45S pre-rRNA,Cleavage(剪切),15,Modification of pre-rRNAs,假尿（嘧啶核）苷,Synthesis and Processing of 5S rRNA,The 5S rRNA genes are located outside the nucleolus(核仁)and have over 50000 copies.Transcription RNA polymerase III:binds to a promoter site(内部启动子)located within the transcribed portion of the gene.,Prokaryotic cells:all rRNA genes(E.coli)as a transcription unit of 16S-23S-5S，7 copies，several types of tRNA molecules lie in every transcription unit simultaneously.Transcription:RNA polymerase,rRNAs purified and ribosomal proteins isolated using ion exchange resin are mixed:-a self-assembly processRibosomal proteins combine with rRNAs in sequence(temporally and spatially)-primary binding protein-secondary binding protein.,(2)Assembly of ribosomal subunits,The relationship between rRNA and ribosomal proteins in small subunit of E.coli ribosome.The lines denote the interaction,which the thick lines represent stronger role,the thin ones represent weaker role（Alberts et al，1989）.,Even if ribosomal proteins(E.coli)are mutated or deleted,there are no effect of“all”or“none”on protein synthesis.If rRNA genes are mutated,protein synthesis will be controlled.If rRNAs are removed,ribosome can not keep in normal appearance rRNAs were structurally central component and proteins play auxiliary(辅助)roles in keep rRNA conformation in ribosome.,The effect of rRNAs and proteins on the role of the ribosome,L11-rRNA复合物的三维结构(引自Porse et.al.,1999),3)Function of Ribosome,23,氨基酸活化,翻译起始,延伸,终止,(1)Relationship between its structure and Function,There are a series of key active sites relative to protein synthesis.Four binding sites for RNA molecules:one is for the mRNA and three for tRNAs.,The binding site for mRNA,澳大利亚科学家约翰夏因与琳达尔加诺,Three binding sites for tRNAs,aminoacyl-tRNA（acceptor site）,peptidyl-tRNA(donor site),spent tRNA-binding site(exit site),A,P,E,A catalytic site for a peptidyl transferase(肽酰转移酶)23S rRNA catalyzes the formation of peptide bond.,Harry Noller等(1992)50S ribosomal proteins,treatment with protease K、SDS、phenol（苯酚）:no effect on protein synthesis 50S rRNA treatment with RNase(核酸酶):inhibiting protein synthesis,23S rRNA forms a highly structured pocket through a network of hydrogen bonds,precisely orients the two reactants and thereby greatly accelerates their covalent joining.The main role of the ribosomal proteins seems to stabilize the RNA core.23S rRNA owns enzyme activity-Ribozyme.Ribozymes:RNA molecules capable of catalyzing chemical reactions in the absence of proteins.,Figure 671 Structure of the L15 protein in the large subunit of the bacterial ribosome.The globular domain of the protein lies on the surface of the ribosome and an extended regionpenetrates deeply into the RNA core of the ribosome.The L15 protein is shown in yellow and a portion of the ribosomalRNA core is shown in red.(From D.Klein,P.B.Moore and T.A.Steitz,J.Mol.Biol.340:141147,2004.,other binding sites The binding sites for translation initiation factors(IF1,IF2,IF3),elongation factors(EF-Tu and EF-Gin bacteria,and EF1 and EF2 in eukaryotes),and release factors(RF1/2,RF3),The binding sites for the antibiotics,(嘌呤霉素),(苯丙氨酰基-tRNA),（苯丙氨酸）,the existence forms of ribosomes in Cytosol,4)Polyribosome(多聚核糖体),Polyribosome(or polysomes):Many ribosomes connected by a single of mRNA molecule-ribosomes spaced as close as 80 nucleotides apart（每隔80核苷酸有一个核糖体）The synthesis of most proteins:between 20 seconds and several minutes,多聚核糖体视频,Biological significance for polyribosomesThe number of the polypeptide molecules synthesized per unit time are roughly equal,regardless of their molecular size or the length of the mRNA.These multiple initiations allow the cell to make more protein molecules in a given time.These multiple initiations improve the utilization efficiency for mRNAs.(一条mRNA可同时合成多条多肽链),35,2.Sorting and targeting of proteins,36,two types of protein translocation ways,(共翻译转运或协同翻译),（翻译后转运）,37,-signal sequence of proteins-sorting receptor in target organelles,(1）Targeting of proteins,38,核定位信号（nuclear-localization signal,NLS）,Gunter Blobel 1999年,39,Gated transport(门控运输)Transmembrane transport(穿（跨）膜运输：胞质中合成的肽链进入线粒体、内质网时，肽链去折叠)Vesicle transport(膜泡运输),(2）Transport pathways of proteins in cytoplasmic matrix,都需能量,Vesicle transport(膜泡运输),Proteins located in different compartments were sorted？,41,41,Known Sorting Signals That Direct Proteins to Specific Transport Vesicles,42,42,1）Types of coated vesicles（衣被小泡）clathrin-coated vesicle(网格（成笼）蛋白有被小泡)COPII-coated vesicles(COPII有被小泡)COPI-coated vesicles(COPI有被小泡),43,43,2)Transport routes of coated vesicles in cells,ER,Golgi,Lysosome,Plasma membrane,COPII,Clathrin,Clathrin,plant vacuole,COPI,endosome,CGN TGN,COPI,Clathrin,Clathrin,Clathrin,Transport routes for different vesicles？,44,44,44,3）Formation of coated vesicles,Clathrin(网格蛋白或笼形蛋白)coated vesicles,-Clathrin:TGN of Golgi complex and plasma membrane.clathrin Dimer(重链和轻链:网格蛋白)clathrin triskelion（包被的结构单位）,Clathrin coated pits,三个Clathrin二聚体,三脚蛋白复合体,Clathrin coated pits,三个Clathrin triskelion(三脚蛋白复合体),Assembly pathway of clathrin coat subunits,46,46,46,（发动蛋白缢断蛋白）,发动蛋白(dynamin)是一种GTPase,一个hsp70家族的伴侣蛋白作为一个脱被的ATPase参与了成笼蛋白的脱被过程,ARF（assembly response factor-装配反应因子：一种单体GTPase）is involved in formation of clathrin coat in Golgi complex,as coat-recruitment GTPase.,衣被成分主要为网格蛋白和衔接蛋白,衔接蛋白识别受体,网格小泡装配视频,47,47,COPI-coated vesicles:,COPI-coated proteins:eight types of subunits.COPI:formation in CGN of Golgi complex.ARF is also involved in formation of COPI coat（similar to clathrin）.,COPII-coated vesicles（最早发现-酵母）,大多数跨膜蛋白是直接结合在COP II衣被上，少数跨膜蛋白和多数可溶性蛋白通过受体与COP II衣被结合,COPII-coated proteins:five types of protein subunits SAR 1 is involved in formation of COPII in ER：Sar-GTP与内质网膜的结合起始COPII亚基的装配,衣被小泡形成的部位，称为内质网出口（exit sites），该处没有核糖体。,49,美国科学家詹姆斯罗思曼、兰迪谢克曼以及德国科学家托马斯祖德霍夫-破解囊泡运输系统的奥秘-细胞的“物流系统”,詹姆斯罗思曼,兰迪谢克曼,托马斯祖德霍夫,【2013诺贝尔奖】生理学奖,三种囊泡介导不同途径的运输，分工井井有条。,3.Fate of proteins,受损伤、衰老失效和折叠错误的蛋白质被降解,蛋白酶体（proteasome),Ubiquitin(泛素):76 amino acid residues,(1)The sites of degradation of proteins in cells-Lysosomes（受损伤蛋白）-proteasomes(蛋白酶体):80%proteins(2)Controlling life of proteins-Vasrhavksy et al(美国麻省理工)-N-end rule(N-末端规律)of controlling life of proteins:amino acid residues at N-end.,细胞的垃圾桶,泛素-蛋白酶体降解途径(ubiquitin proteasome pathway)是一个精细控制的过程,泛素酶负责活化泛素,泛素结合待降解的靶蛋白的赖氨酸侧链,蛋白酶体“帽”识别已泛素化的蛋白,泛素激活酶E1,泛素缀合酶E2,泛素连接酶E3,靶蛋白的赖氨酸侧链与泛素形成肽键,53,思考题,1、比较真核和原核胞质核糖体的结构和化学组成。2、核糖体是否只是细胞合成蛋白的场所？3、比较ribosome和polyribosome的差异，并简述多聚核糖体的生物学意义。4、何为Ribozyme？5、区分翻译共转运和翻译后转运的概念。6、简述在细胞质基质中合成的蛋白，其合成后有几种转运方式，并比较分析细胞核蛋白、细胞外基质蛋白和溶酶体酶在胞质中合成、转运至靶位点的不同之处。7、简述膜泡运输中三种常见小泡的类型，并比较它们运输途径的差异。8、简述蛋白质的降解方式。,