细胞生物学课件：第6章 细胞质核糖体

1、1CHAPTER 6 Cytoplasm , Ribosome and RNAs2OUTLINE6.1 Structure and function of cytoplasm6.2 Ribosome6.3 Ribozyme and Non-coding RNAsCytosol: dissolved nutrients and waste productsdissolved macromolecules, such as RNAs and proteinsmany salts OrganellesCytoskeletonCytoplasmic inclusions6.1.1 Structure

2、of cytoplasmOrganelles (1) nucleolus; (2) nucleus; (3)ribosomes; (4) vesicle; (5) rough endoplasmic reticulum (ER); (6) Golgi apparatus;(7)cytoskeleton; (8)smoothER;(9)mitochondria; (10) vacuole; (11) cytosol; (12) lysosome; (13) centrioles within centrosome Cytoplasmic inclusions crystals of calciu

3、m oxalate or silicon dioxide in plants (草酸钙或二氧化硅) granules of starch, glycogen, or polyhydroxybutyrate (聚羟基丁酸酯), which can store energy. Lipid droplets, storing fatty acids and sterolsMetabolic pathways and biochemical reactionsUbiquitin-proteasome systemFunctions of organellesMaintenance and moveme

4、ntStorage 6.1.2 Functions of cytoplasm76.2 Ribosome8核糖体(ribosome)The ribosome is a ribonucleoprotein particle (核糖核蛋白颗粒) composed of RNA and protein，and serves as the site of protein synthesis. 核糖体的主要成分ribosome RNA (rRNA)：60% ribosome protein： 40%6.2.1 Ribosome structure 9Ribosome subunits10Types of

5、RibosomesTypeProkaryotesEukaryotessediments70S80SMolecular weight2.5x103 kD3.94.5x103 kDsubunits50S and 30S60S and 40SAbundance15102-18103/cell106-107 /cellotherpolyribosome or polysomefree ribosomemembrane-bound polyribosome or polysome11Chemical Composition of Ribosome12Compositions of ribosome fr

6、om 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 Mitochon

7、drion 78S 60S (大亚基) 26S(Plant) 45S (小亚基) 18S, 5SChloroplast 70S 50S (大亚基) 23S 30S (小亚基) 16S, 5S13Mg2+ and subunit assembly in ribosome 70S核糖体在Mg2+的浓度小于1mM的溶液中易解离; 当Mg2+ 浓度大于10mM, 两个核糖体通常形成100S的二聚体。 146.2.2 Biogenesis of Ribosomeself assembly in cells with different procedures in prokaryotes and euka

8、ryote.Synthesis of ribosome proteins and rRNAsAssembly of ribosome subunits 15Ribosome genesrRNA gene amplificationmulti-copies of rRNA gene on chromosomes 7 copies of rRNA genes in E.coli; 1001000 copies of 18S、 5.8S and 28S rRNA genes in eukaryotes, 50000 copies of 5S rRNA gene. 1618S, 5.8S and 28

9、S rRNA gene transcription and processing in eukaryotes rDNA orgnization on chromosomes18S, 5.8S and 28S rRNA genes on one chromosome whereas 5S rRNA genes on another huma rRNA gene orgnizationeach group of rRNA genes have 200 copies in a cluster and each copy as a rDNA transcription unit.rRNA genes

10、located on human 13, 14, 15, 21, 22 chromosomes and assembly as nucleolus during interphase.17 transcription45S primary transcript ;pre-rRNA: 34S45S;RNA polymerase I is specifically devoted to transcription of the three largest species of rRNAs;18 pre-rRNA in different species Species pre-rRNA Droso

11、phila 34S S. pombe 37S Tobacco 38S Frog 40S Chicken 45S Mouse 45S Human 45S19The rRNA transcription unit20The synthesis of rRNAnucleoliTranscription of rDNA21Processing of 45S pre-rRNA22前体rRNA被甲基化的部位一直保持到成熟的rRNA中;人为地阻断前体rRNA的甲基化，前体rRNA的加工也被阻断;推测前体rRNA的甲基化对rRNA的加工具有指导作用。Methylation of rRNA23Synthesis

12、 and Processing of the 5S rRNA In eukaryotes, the 5S rRNA molecules are encoded by a large number of identical genes that are separate from the other rRNA genes and are located outside the nucleolus. The 5S rRNA genes are transcribed by RNA polymerase III. The 5 end of the primary transcript is iden

13、tical with that of the mature 5S rRNA, but the 3 end usually contains extra nucleotides that are removed during processing. 24RNA polymerase III is unusual among the three polymerases, it binds to a promoter site (内部启动子) located within the transcribed portion of the gene rather than a site upstream

14、from the genes 5 flank. 25原核生物rRNA基因原核与真核生物的rRNA基因在组织结构上的差异:重复频率：如E.coli，只重复了7次；细菌的5S rRNA与16SrRNA 、23S rRNA基因组成一个转录单位，在染色体上的排列顺序是16S-23S-5S。核酸酶RNase将16S-23S-5S rRNA前体切割成单体;细菌中的rRNA转录单位除了三种rRNA外，同时有几种tRNA分子。26原核生物 Pre-rRNA的加工27Assembly of ribosome in prokaryotes：self-assembly: protein and rRNA assem

15、bled sequentially (temporally and spatially); some protein has to bind to rRNA first and then other proteins28Assembly of small subunit of ribosome in E. Coli29初级结合蛋白(primary binding protein)直接同rRNA结合, 其中同16S rRNA结合的初级蛋白有14种, 它们是: S3, S4, S17, S20, S6, S15, S8, S18, S9, S11, S12, S13, S7, S1。 同5S rR

16、NA结合的有11种。次级结合蛋白(secondary binding protein)不直接同rRNA结合, 而是同初级结合蛋结合。它们是: S10, S16, S2, S6, S21, S14, S19。根据同rRNA结合的顺序,将核糖体蛋白分为两种:30核糖体结合蛋白31 Assembly of ribosome in eukaryotes80S前体颗粒形成 45S rRNA+5S rRNA+蛋白质 45S rRNA 41S rRNA大小亚基前体形成: 大:32S rRNA和5S rRNA 小:20S的前体rRNA；小亚基成熟与运送: 20S rRNA18S rRNA大亚基成熟与运送 32

17、S rRNA28S rRNA+5.8S rRNA?32Ribosome assembly in human cells336.2.3 Function of ribosomesRNA binding sites:binds to SD sequence of mRNA:蛋白质合成中各位点 (E, P, A sites) 的协同性蛋白质合成的基本过程多聚核糖体(polysome / polyribosome)蛋白质合成的抑制剂蛋白质寿命34Functional sites on ribosomes35SD (Shine-Delgarno) sequenceAminoacyl-tRNA37Anti

18、code of tRNA: A, U, C, G + Inosine 肌苷，(次黄嘌呤 hypoxanthine)39Start of protein synthesis30S+mRNAAminoacyl-tRNA+ Large Unit完整起始复合物Figure 6-72 (part 1 of 5) Molecular Biology of the Cell ( Garland Science 2008)Figure 6-72 (part 2 of 5) Molecular Biology of the Cell ( Garland Science 2008)IFs or eIFsFigur

19、e 6-72 (part 3 of 5) Molecular Biology of the Cell ( Garland Science 2008)Figure 6-67 Molecular Biology of the Cell ( Garland Science 2008)Figure 6-72 (part 4 of 5) Molecular Biology of the Cell ( Garland Science 2008)Figure 6-72 (part 5 of 5) Molecular Biology of the Cell ( Garland Science 2008)Fig

20、ure 6-66 (part 4 of 4) Molecular Biology of the Cell ( Garland Science 2008)Figure 6-74 (part 1 of 3) Molecular Biology of the Cell ( Garland Science 2008)Termination and releaseFigure 6-74 (part 2 of 3) Molecular Biology of the Cell ( Garland Science 2008)Figure 6-74 (part 3 of 3) Molecular Biology

21、 of the Cell ( Garland Science 2008)51多聚核糖体?52嘌呤霉素对蛋白质合成的抑制作用53嘌呤霉素对蛋白质合成的抑制作用Figure 6-79 Molecular Biology of the Cell ( Garland Science 2008)Table 6-4 Molecular Biology of the Cell ( Garland Science 2008)Figure 6-84 Molecular Biology of the Cell ( Garland Science 2008)Figure 6-85 Molecular Biology

22、 of the Cell ( Garland Science 2008)Figure 6-86 Molecular Biology of the Cell ( Garland Science 2008)Figure 6-88 Molecular Biology of the Cell ( Garland Science 2008)606.3 Ribozyme and Non-coding RNARibozymes :A ribozyme is defined as an RNA molecule capable of catalyzing a chemical reaction in the

23、absence of proteins.Deoxyribozymes:Deoxyribozymes or DNA enzymes or catalytic DNA, rDNAzymes are artificially DNA molecules that have the ability to perform a chemical reaction, such as catalytic action.Rz与DRz的比较：特性DRzRz化学组分DNARNA催化特性高度专一性和高效性，与底物严格按照碱基配对原则结合。DRz-mRNA杂交分子较Rz-mRNA杂交分子易解离；催化效率远远高于Rz 高

24、度专一性和高效性，与底物严格按照碱基配对原则结合 成构及活性中心特点缺少2-羟基，分子量较小，受靶序列二级结构的影响较弱，活性中心结构有更大的选择性，对底物的趋近性比Rz好，一般DRz较Rz有更多的剪切靶位可供选择 每个核苷酸都具有一个2-羟基，结构相对复杂 底物DNA或mRNAmRNA稳定性在生理pH、温度和离子强度等条件下，稳定性约为RNA的105倍；在细胞培养和体内环境中，对核酸酶的降解作用比Rz高得多 与DRz相比稳定性差 62types of RzRNA and protein complexRibonuclease P (核糖核酸酶 P): tRNA (375bp)+ protei

25、n (20kDa), type introncatalytic small molecule RNA: no protein requiredPeptidyl transferase 23S rRNARNase PSpliceosomesLeadzyme Hammerhead ribozyme64Rz activity of 23S rRNA in 50S large ribosome subunit肽酰转移酶 (peptidyl transferase)Harry Noller in 1992实验过程: 破坏50S亚基核糖体蛋白质 蛋白酶K、SDS、苯酚等 破坏rRNA 核酸酶处理50S亚基

26、核糖体 抑制蛋白质合成 用抑制剂处理结论: 具有肽酰转移酶的活性分子是23SrRNA。self-splicing RNAs66 Splicing MechanismsNuclear splicingGroup I intron splicingGroup II intron splicing67核剪接(nuclear splicing)主要对hnRNA中切除内含子剪接发生在细胞核中RNA剪接(RNA splicing)即从pre-mRNA中切除内含子将外显子连接成成熟的mRNA68Nuclear splicing Mechanisms GU-AG rule；Spliceosomes : Com

27、posed of proteins and RNAs. The RNA components of the spliceosome are five types of small nuclear RNAs (snRNAs) called U1, U2, U4, U5, and U6. Lariat-like structure 69GU-AG rule70核剪接71Group I intron splicing结构特点: 内含子转录后可以形成9个由碱基 配对形成的特定二级结构, 分别命 名为P1至P9, P1和P7是保守的。需要游离的鸟苷存在的细胞器:核、线粒体、叶绿体基因:前体rRNA、mR

28、NA和tRNA72Group I intron splicing73Group II intron splicing结构特点: 内含子转录后可以形成6个发夹环GU-AG规则不需snRNA参与,不形成剪接体形成套索存在的细胞器:线粒体、叶绿体基因:前体mRNA74II组内含子的剪接75RNA剪接的三种途径?外源鸟苷内源腺苷lariat cleaving Rzs RNase PSpliceosomes77RNA editing RNA editing refers to RNA processing events (other than splicing) that alter the prote

29、in-coding sequences of some mRNAs. This unexpected form of RNA processing was first discovered in mitochondrial mRNAs of trypanosomes(锥虫), in which U residues are added and deleted at multiple sites along the molecule. More recently, editing has also been described in mitochondrial mRNAs of other or

30、ganisms, chloroplast mRNAs of higher plants, and nuclear mRNAs of some mammalian genes.78Editing in mammalian nuclear mRNAs, as well as in mitochondrial and chloroplast RNAs of higher plants, involves single base changes as a result of base modification reactions;RNA editing reactions include the deamination of cytosine to uridine and of adenosine to inosine(次黄苷);Apo-B48(2152 amino acids) is synthesized in the intestine as a result of translation of an edited mRNA in which a C has be