细胞生物学：6 Cytoplasm and Ribosome-2

1the existence forms of ribosomes in CytosolProkaryotesEukaryotes70S80Spolyribosome or polysomefree ribosome,polysome,membrane-bound polyribosome 24)Polyribosome(多聚核糖体多聚核糖体)Polyribosome(or polysomes):A cluster of ribosomes connected by a single of mRNA molecule more effectively synthesize proteins,in which ribosomes spaced as close as 80 nucleotides apart.The synthesis of most protein molecules:between 20 seconds and several minutes 3Biological significance for polyribosomesFor synthesis of a variety of intracellular polypeptides:the 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.43.RIBOZYME(核酶核酶)1)Discovery of ribozyme(Rz)-1981,Thomas Cech等,tetrahymena(四膜虫)-pre-26S rRNA processingintron(413nt)cleavage(剪切)-pre-26S rRNA-self-splicing(自剪接)-pre-mRNA,tRNA or rRNA processing of prokaryotes,eukaryotes,mitochondrion or chloroplst-self-splicingnRibozymes(molecular scissors):RNA molecules capable of catalyzing chemical reactions in the absence of proteins.5n 1989,Thomas Cech obtained the nobel prize in chemistry for discovery of ribozyme-The classic concept that enzymes are proteins was broken for the discovery of ribozyme.1994,discovery of deoxyribozymes(脱氧核酶)Deoxyribozymes(DRz,DNA enzymes or catalytic DNA):artificially single-stranded DNA molecules capable of catalyzing chemical reactions in the absence of proteins.Compared with proteases,Both DRz and Rz have lower catalytic efficiency in catalyzing chemical reactions.62)Type of ribozymes in organisms(1)RNA and protein complex:-Ribonuclease P(核糖核酸酶核糖核酸酶 P):375nt ssRNA and 119-aa polypeptide),pre-tRNA introns processing,Prokaryotes and eukaryotes(nucleus,mitochondria and chloroplasts).-Spliceosome(pre-mRNA,proteins and snRNA):A splicing intermediates formed in the pre-mRNA(hnRNA）processing in the nucleus-Nuclear splicing(核剪接核剪接).7 RNase P-cleavageSpliceosomesself-cleavagepcleaving RNAs(cleaving Rzs)水解酶水解酶8(2)Type I and II intron:pre-mRNA introns splicing，Self-Splicing RNAscatalytic small molecule RNA:no protein required9(3)Small molecule RNA(substrate and enzyme)：self-cleavage within molecules-Hammerhead ribozyme(锤头型核酶:plant virus),-HDV Rz(丁型肝炎病毒核酶)Common characteristic：cleave or ligate phosphodiester bonds(磷酸二酯键)；substrate:RNA;Difference:the substrate(own RNA or other RNA(4)Peptidyl transferase(23S rRNA)锤头型核酶1010RNA editing(RNA编辑编辑)：RNA processing events such as insertion,deletion or replacement of bases which alter the protein-coding sequences of some mRNAs.mitochondrial mRNAs of other organisms,chloroplast mRNAs of higher plants,and nuclear mRNAs of some mammalian genes.谷氨酰胺谷氨酰胺Apolipoprotein B(apoB,人的人的载脂载脂蛋白蛋白)intestineliver11 The mechanism of RNA editing guide RNAs(gRNAs:指导指导RNA)-about 60-80 nt，containing a 3-oligoU tail and a sequence in the middle precisely complementary to the edited mRNA，and an anchor sequence at the 5 end,complementary to the sequence of mRNA pre-edited.editosome(编辑体编辑体):an editing complex forming in the editing process,the transcripts corrected by the internal sequence of gRNA as a template and producing mRNA editedU donor：U of 3-oligoU tail1212gRNA指导锥虫线粒体指导锥虫线粒体细胞色素氧化酶亚基细胞色素氧化酶亚基基因转录基因转录物的编辑（物的编辑（Nelson等等2000）（）（a）4个个U的插入；（的插入；（b）编）编辑过程中的辑过程中的gRNA模板作用模板作用134)Application of Rz（1）Research on the origins of life on EarthIn the process of biological evolution,RNA could be the biological macromolecule that first appears on the earth because of the discovery that Rz can catalyze RNA duplication 14（2）Prevention of plant virus Artificially design and synthese monovalent or divalent ribozymes-catalyze specific RNA cleavage-decrease mRNA level of plant virus（3）Treatment of human diseases Hepatitis(肝炎)：hairpin Rz-Inhibition of HBV(hepatitis B virus,乙肝病毒)replication;Hammerhead ribozyme-Inhibition of HCV(hepatitis C Virus,丙型肝炎病毒)AIDs Cancer154.Small RNAs(小小RNA)in eukaryote size:100-300nt,RNA polymerase II or Type:-snRNA(small nuclear RNA:pre-mRNA processing and rRNA processing(核仁核仁):small nuclear ribonucleoproteins,rich uridine，called U1，U2.)-scRNA(small cytoplasmic RNA)-some of small RNAs:2024nt ssRNA,as a negative regulatory factor，controlling target mRNA expression at the level of transcription or translation,such as miRNA(microRNA:translation repression),siRNA(small interfering RNA:target mRNA cleavage)161)Discovery of miRNA-VictorAmvros等(1993)-Inhibition of larval(幼虫)development(lin-14 down-regulation)in C.elegans(线虫)-microRNA from lin-4(NON-CODING RNA)-2000let-7(microRNA)development control -2001small RNAs were named miRNAs -2002clone Arabidopsis miRNA -miRNAs in many species -Up to now,nearly twenty thousand miRNAs from more than one hundred species log on miRNA data bank(http:/www.Mirbase.org/)of Sanger Institute.172)generation of miRNAmiRNA-Hundreds of nucleotide pri-miRNA from genome by RNA polymerase II-pri-miRNA cut by Dicer:pre-miRNA with a stem-loop structure-Animals:pre-miRNA exported by Exportin 5 to cytoplasm and cut by Dicer-miRNA：miRNA-Plants:pre-miRNA cut by DCL-dimer of miRNA：miRNA(n)-dimer exported by HASTY from nucleus to cytoplasm-mature miRNA released by helicase(解旋酶解旋酶)Dicer-like 118 generation of siRNA-Endogenous or exogenous RNA(invading virus)-Small dsRNA(21nt-25nt)cut by Dicer(DCL4 in plants)-mature ssRNA植物病毒入侵诱导初生siRNADicer切割切割作用于其来源靶作用于其来源靶mRNA，降解降解mRNA193)Mechanism of miRNA RISC(RNA induced silencing complex):Dicer(RNase domain:keep the RISC intermediate stable during the process of RISC assembly),Argonaute(enzyme activity center of cleavage),miRNA(binding to target mRNA through complementation:repressing protein translation by preventing the ribosome moving on mRNA)20思考题思考题1 1、简述细胞质的概念及组成成分、简述细胞质的概念及组成成分2 2、比较真核和原核细胞核糖体的结构和化学组成、比较真核和原核细胞核糖体的结构和化学组成3 3、核糖体是否只是细胞合成蛋白的场所？为什么？、核糖体是否只是细胞合成蛋白的场所？为什么？4 4、比较、比较ribosomeribosome和和polyribosomepolyribosome的差异，并简述多聚的差异，并简述多聚核糖体的生物学意义核糖体的生物学意义5 5、何为、何为RibozymeRibozyme和和DeoxyribozymeDeoxyribozyme，列出细胞中常见，列出细胞中常见的核酶并简述核酶的应用前景的核酶并简述核酶的应用前景6 6、小、小RNARNA有哪些，查找相关资料了解其研究进展及应有哪些，查找相关资料了解其研究进展及应用用219.Endomembrane System22The secretory pathway:pancreatic tissue:culturing in the culture medium adding amino acids by isotope labeling pulse-chase experiment and Autoradiography techniques23Subcellular fractionation Microsome(微粒体微粒体)：ER fragments reseal to form small closed vesicles-microsomes.The microsomes are almost derived from ER and still capable of protein synthesis and translocation,protein glycosylation,Ca2+uptake and release,and lipid synthesis.Isolation of microsomes24yeast mutants relative with endomembrane functionsMutations25OUTLINEBasic concepts of Endomembrane System Component of Endomembrane System Endoplasmic Reticulum（ER）Golgi complex(GC)LysosomesCharacteristics of endomembrane systemBiological significance of endomembrane systems261.The concepts and component of endomembrane System:a l l m e m b r a n e-e n c l o s e d compartments in the cytosol,which are correlative in membrane structure,function and biogenesis,including Nucleus,ER,Golgi complex(GC),lysosomes,Vacuoles(endosomes-胞内体胞内体)and secretory vesicles(分泌泡分泌泡).27Membrane-bounded organelles:all organelles with membrane structure in the cytosol.细胞膜、生物膜、细胞内膜系统281)Endoplasmic Reticulum（ER）ER discovery：1945-K.R.Porter et al.cultured mouse fibroblast(成纤维细胞成纤维细胞):a netlike labyrinth(迷宫迷宫)throughout the entire cytosol.29(1)(1)Structure ER is a network of membrane-enclosed branching tubules and flattened sacs that extends from the nuclear membrane throughout the cytoplasm.cytosolic space(胞质胞质溶胶面溶胶面)(潴泡面潴泡面)30 ER is the largest organelle in most eukaryotic cells.ER cisternal space often occupies about 10%of the total cell volume.31ER membrane may account for about half of the total area of all cell membranes.32(2)(2)Types of endoplasmic reticulum Rough endoplasmic reticulum(R E R)-f l a t t e n e d s a c s,membrane-bound ribosomes bound to its cytosolic surface.Smooth endoplasmic reticulum(SER)-branching tubules,no ribosomes.33Special types of ER microsome (微粒体微粒体)sarcoplasmic reticulum(心肌和骨骼肌心肌和骨骼肌:肌质网肌质网)34(3)(3)Chemical component of ER ER:proteins(60%),Lipids(40%).Proteins:the distribution of ER proteins is heterogeneous(异质性异质性)glucose-6-phosphatasemarker enzyme ribophorin(核糖体结合糖蛋白):RER P450酶系:SER translocon（易位子）protein complex:RERLipids：Phospholipids(Principal lipid ingredient)