分子生物学-蛋白质合成.ppt

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1、Chapter 9Protein synthesisCoding region编码区 is a part of the gene that represents a protein sequence.Coding strand 编码链of DNA has the same sequence as mRNA.template strand模板链 of double-stranded DNA is the one that is used to specify the sequence of a complementary single strand of RNA.(The non-templat

2、e strand is identical in sequence to the RNA product.)Transcription 转录is synthesis of RNA on a DNA template.Translation 翻译is synthesis of protein on the mRNA template.IntroductionTranscription generates an Transcription generates an RNA which is RNA which is complementary to the DNA complementary to

3、 the DNA template strand and has the template strand and has the same sequence as the DNA same sequence as the DNA coding strand.Translation coding strand.Translation reads each triplet of bases reads each triplet of bases into one amino acid.Three into one amino acid.Three turns of the DNA double t

4、urns of the DNA double helix contain 30 bp,which helix contain 30 bp,which cide for 10 amino acids.cide for 10 amino acids.Aminoacyl氨酰-tRNA is transfer RNA carrying an amino acid;the covalent 共价linkage is between the NH2 group of the amino acid and either the 3-or 2-OH group of the terminal base of

5、the tRNA.Aminoacyl-tRNA synthetases are enzymes responsible for covalently linking amino acids to the 2-or 3-OH position of tRNA.Anticodon反密码子 is a trinucleotide三核苷酸sequence in tRNA which is complementary to the codon in mRNA and enables the tRNA to place the appropriate amino acid in response to th

6、e codon.Loop 环is a single-stranded region of a hairpin发夹 in RNA(or single-stranded DNA);corresponds to the sequence between inverted repeats 反向重复in duplex DNA.Stem is the base-paired segment of a hairpin.Transfer RNA is the adapterA tRNA has the dual A tRNA has the dual properties of an adaptor prop

7、erties of an adaptor that recognizes both the that recognizes both the amino acidamino acid and and codoncodon.The.The adenosineadenosine腺苷腺苷at at 3 end is 3 end is covalently linked to an covalently linked to an amino acid.The amino acid.The anticodon anticodon basebase pairs with the codon pairs w

8、ith the codon on mRNA.on mRNA.The tRNA cloverleafThe tRNA cloverleaf三叶草结构三叶草结构has has invariantinvariant不变的不变的 and and semi-invariant bases,semi-invariant bases,and a conserved and a conserved 保守保守的的set of base pairing set of base pairing interactions.interactions.Transfer RNA folds into Transfer RN

9、A folds into a compact L-shaped a compact L-shaped tertiary structure tertiary structure 三级结三级结构构with the amino acid at with the amino acid at one end and the one end and the anticodon at the other anticodon at the other end.end.A space-filling model A space-filling model shows that tRNA tertiary sh

10、ows that tRNA tertiary structure is compactstructure is compact紧密的紧密的.The two views of tRNA The two views of tRNA are rotated by 90are rotated by 90o o.Photograph kindly Photograph kindly provided by S.H.Kim.provided by S.H.Kim.The meaning of tRNA is determined by its The meaning of tRNA is determin

11、ed by its anticodon and not by its amino acid.anticodon and not by its amino acid.A ribosome consists of two subunits亚基.Messenger RNA is translated by ribosomes 核糖体A polyribosomeA polyribosome多核糖体多核糖体consists of consists of an mRNAan mRNA being being translated simultaneously by translated simultane

12、ously by several ribosomesseveral ribosomes moving in the moving in the direction from 5-3.Each ribosome has direction from 5-3.Each ribosome has two tRNAtwo tRNA molecules,molecules,one carrying the nascent amino acidone carrying the nascent amino acid初生氨基酸初生氨基酸,the second,the second carrying the n

13、ext amino acid to be added carrying the next amino acid to be added 添加氨基酸添加氨基酸.A ribosome assemblesA ribosome assembles装配装配 from its subunits on mRNA,from its subunits on mRNA,translates the nucleotide triplets into protein,and then translates the nucleotide triplets into protein,and then dissociate

14、s dissociates 分离分离from the mRNA.from the mRNA.Protein synthesis occurs on polyProtein synthesis occurs on polyriboribosomes.somes.多核糖体多核糖体Messenger RNA is translated by ribosomes that cycle through a pool.Considering E.coli in terms of its macromolecular components大分子成分.mRNA is transcribed,translate

15、d,mRNA is transcribed,translated,and degraded simultaneously and degraded simultaneously 同同时时in bacteria.in bacteria.细菌中转录与翻译同时进行转录开始后核糖体附着在mRNA的5端开始翻译同时，mRNA沿从5至3的方向迅速降解在37C，mRNA转录速度约为40核苷酸秒。蛋白质合成速度约为15氨基酸秒。转录并翻译180KD蛋白质的5000hp的 mRNA需时 2分钟Transcription units can be visualized in bacteria.Bacterial

16、mRNA includes non-translated as well as translated Bacterial mRNA includes non-translated as well as translated regions.Each coding region has its own initiation and termination regions.Each coding region has its own initiation and termination signals.A typical mRNA may have several coding regions.s

17、ignals.A typical mRNA may have several coding regions.大多大多数细菌基因通过多顺反子进行表达数细菌基因通过多顺反子进行表达位于编码区之前的5端附加序列，叫做前导序列终止信号后组成3末端的附加序列叫做跟踪序列。顺反子间区Eukaryotic mRNA is modified by addition of a cap to the 5 end and poly(A)to the 3 end.Translation of eukaryotic mRNA expression of mRNA in animal cells requires tra

18、nscription,modification,processing,nucleocytoplasmic transport,and translation mRNAmRNA的表达需转录、修饰、加工、的表达需转录、修饰、加工、核质转运和翻译核质转运和翻译.真核生物mRNA只占整个细胞RNA的一小部分（质量的3）。然而相对稳定。典型动物中mRNA的半衰期范围是424小时Exogenous mRNAs can be translated by cell-free systems Exogenous mRNAs can be translated by cell-free systems or by

19、 injection into or by injection into XenopusXenopus oocytes oocytes非洲爪蟾卵母细胞非洲爪蟾卵母细胞.恢复 cellfree系统包括核糖体、蛋白质因子和 tRNA。1、转录开始于核苷三磷酸（通常是膘呤，A或G）2、转录物的起始序列：5pppA(G)pNpNpNpNp 3、鸟苷酸转移酶催化5端添加甲基化的G，即RNA帽子，根据甲基化的位置和数目不同可分为帽子0、帽子1、帽子2等多种类型4、在真核生物中，每个mRNA分子都是戴帽的。不同类型帽子的比例是一种生物的特征。5、除了戴帽作用中的甲基化外，mRNA中的内部也存在低频率的甲基化

20、作用The 5 end of eukaryotic mRNA is capped帽子的种类b帽子0：出现在所有真核生物中，在末端乌瞟吟的7位上添加一个甲基构成。担负这种修饰作用的酶是细胞质中的乌瞟鸣一7一甲基转移酶。b帽子1：具有两个甲基团。再在转录物中第1个碱基2O位置上添加一个甲基，由2一甲基一转移酶催化。是除单细胞生物以外所有其他真核生物中都有的最主要的帽子。在少量较高等真核生物中，当2O位置上已有甲基时，可将甲基团添加到该碱基的N6位。b帽子2：有些物种，甲基基团可以添加到戴帽mRNA的第3碱基(转录物中第2个碱基)的2O位置。这种帽子具有三个甲基团，通常低于戴帽群体总量的1015。帽

21、子结构的作用b对mRNA的翻译活性很重要，可使之免受外切核酸酶的降解b对mRNA与核糖体的结合包括起始区的辨认也很重要b与蛋白质翻译的起始有关，没有帽子结构不能与核糖体的小亚基形成复合物The cap blocks the 5 end of mRNA and may be methylated at several positions.cDNA is a single-stranded DNA complementary to an RNA,synthesized from it by reverse transcription in vitro体外.The 3 terminus is pol

22、yadenylatedPoly(A)+RNA can be separated from other RNAs by fractionation on Sepharose琼脂糖(凝胶)-oligo(dT).多聚A尾的作用b不是所有的mRNA都有多聚A尾，如组蛋白mRNA的3端有特殊的二级结构，没有多聚尾b多聚A尾对mRNA的稳定性有一定作用，即与mRNA的降解有关。在多聚A尾上存在一些多聚A结合蛋白，可能与mRNA的稳定性有关。b与mRNA通过核膜进入细胞质有关b与翻译调控有关b可利用poly(A)结构在dT分离柱上通过馏分将mRNA与其它RNA分开。Degradosome降解复合体 is a

23、 complex of bacterial enzymes,including RNAases,a helicase,and enolase磷酸丙酮酸水合酶(a glycolytic糖酵解enzyme),which may be involved in degrading mRNA.Bacterial mRNA degradation involves multiple enzymesDegradation of bacterial Degradation of bacterial mRNA is a two stage mRNA is a two stage process.process.

24、EndonucleolyticEndonucleolytic cleavages proceed 5-3 cleavages proceed 5-3 behind the ribosomes.The behind the ribosomes.The released fragments are released fragments are degraded by degraded by exonucleasesexonucleases that move 3-5that move 3-5Exosome外切体 is a complex of several exonucleases involv

25、ed in degrading RNA.Yeast mRNA degradation involves multiple activitiesDegradation of yeast mRNA requires deadenylation脱腺苷,decapping去帽,and exonucleolysis外切核酸酶降解.An ARE in a 3 nontranslated region initiates degradation of mRNA.Sequence elements may destabilize 使使不稳定不稳定mRNAARE:AU-rich sequenceAn IRE i

26、n a 3 nontranslated region controls mRNA stability.转铁蛋白mRNAIRE:Iron regulated elementSurveillance systems check nucleic acids for validity正确性.The term is used in several different contexts.One example is the system that degrades mRNAs that have nonsense mutations.Another is the set of systems that r

27、eact to damage in the double helix.The common feature is that the system recognizes an invalid sequence or structure and triggers a response.Nonsense mutations trigger a surveillance监管监管 systemNonsense mutations may cause mRNA to be degraded.1、DNA携带的遗传信息通过两种途径来表达：DNA转录成mRNA，mRNA翻译成蛋白质。信使RNA由DNA的一条链(

28、模板链)转录而来，与这条链互补，与另外一条链(编码链)一致mRNA的序列，在三联密码子中从5到3，是与蛋白质的N-C端的序列相关的。2、解释密码子意义的是有致密L形三级结构的tRNA，tRNA的一端有反密码子与密码子互补，另一端可以共价地连接特定的与靶密码子一致的氨基酸。一个携带氨基酸的tRNA叫作氨酰基-tRNA。Summary b3、核糖体提供了氨酰基tRNA连接到mRNA密码子上的环境。核糖体小亚基连接mRNA，大亚基携带新生的多肽。核糖体从5端的起始位点向3端的终止位点移动，氨酰基tRNA对应于它们的密码子，这样，延伸的多肽链每经历一个密码子增加一个残基。b4、转录机器对于组织和器官不

29、是特定的；一个来源的mRNA可以被另外一个来源的tRNA和核糖体所翻译。mRNA被翻译的次数决定于起始点与核糖体亲和力及mRNA的稳定性。在许多情况下，mRNA的翻译群体或单独的mRNA被特异地阻止：这被称作翻译控制。b5、一条典型的mRNA包括非翻译的5前导区和3尾及编码区。细菌的mRNA通常是多顺反子，在顺反子间有非翻译区。每个顺反子代表由特定位点起始和终止位点结束的编码区。核糖体亚基连接起始位点，在每一个编码区的终止位点分开。b6、许多核糖体可以同时翻译一条mRNA，生成一个多聚核糖体（或多聚体）。细菌的多聚体很大，典型情况下，一个mRNA上连接有10个核糖体。真核生物多聚体是较小的，通

30、常少于10个，且每个mRNA仅带有一个编码序列。b7、细菌mRNA的半衰期相当短，仅几分钟。基至当下游序列刚被转录5端就已经开始翻译了。降解是由内切酶引起，在特定位点切割，并以从5到3的顺序紧跟在核糖体后面。在这之后，外切酶从释放的3端到5端降解碎片，使之还原为核苷酸。在细菌mRNA中，单独的序列可能会加速或减缓降解。真核生物mRNA可稳定存在几个小时，它们可能有几个序列引发降解。8、真核生物mRNA在它转移到细胞质翻译之前必需在核内被加工，甲基化帽扣在5端。鸟苷酸转移酶催化5端添加甲基化的G，根据甲基化的位置和数目不同可分为帽子0、帽子1、帽子2等多种类型。大多数真核mRNA转录之后在核中有200bp的poly(A)序列添到它的3端，但poly(A)-mRNA被翻译和被降解好像与poly(A)+动力学历程相同。真核细菌mRNA以核糖核蛋白颗粒存在；在许多情况下不翻译的mRNPs被贮藏起来。