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1、Chapter 9,Protein synthesis,Coding 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 plementary single strand of RNA.(The non-template
2、 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.,Introduction,Transcription generates an RNA which is plementary to the DNA template strand and has the same sequence as the DNA codin
3、g strand.Translation reads each triplet of bases into one amino acid.Three turns of the DNA double helix contain 30 bp,which 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 gro
4、up of the terminal base of 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 plementary to the codon in mRNA and enables the tRNA to place the appropriate amin
5、o acid in response to the 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 adapter,A tRNA has the dual properties of an adapto
6、r that recognizes both the amino acid and codon.The adenosine腺苷at 3 end is covalently linked to an amino acid.The anticodon base pairs with the codon on mRNA.,The tRNA cloverleaf三叶草结构has invariant不变的 and semi-invariant bases,and a conserved 保守的set of base pairing interactions.,Transfer RNA folds int
7、o a pact L-shaped tertiary structure 三级结构with the amino acid at one end and the anticodon at the other end.,A space-filling model shows that tRNA tertiary structure is pact紧密的.The two views of tRNA are rotated by 90o.Photograph kindly provided by S.H.Kim.,The meaning of tRNA is determined by its ant
8、icodon and not by its amino acid.,A ribosome consists of two subunits亚基.,Messenger RNA is translated by ribosomes 核糖体,A polyribosome多核糖体consists of an mRNA being translated simultaneously by several ribosomes moving in the direction from 5-3.Each ribosome has two tRNA molecules,one carrying the nasc
9、ent amino acid初生氨基酸,the second carrying the next amino acid to be added 添加氨基酸.,A ribosome assembles装配 from its subunits on mRNA,translates the nucleotide triplets into protein,and then dissociates 分离from the mRNA.,Protein synthesis occurs on polyribosomes.多核糖体,Messenger RNA is translated by ribosome
10、s that cycle through a pool.,Considering E.coli in terms of its macromolecular ponents大分子成分.,mRNA is transcribed,translated,and degraded simultaneously 同时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,Transc
11、ription units can be visualized in bacteria.,Bacterial mRNA includes non-translated as well as translated regions.Each coding region has its own initiation and termination signals.A typical mRNA may have several coding regions.大多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,Euka
12、ryotic 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 transcription,modification,processing,nucleocytoplasmic transport,and translation mRNA的表达需转录、修饰、加工、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
13、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 or by injection into Xenopus oocytes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
14、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作用,The 5 end of eukaryotic mRNA is capped,帽子的种类,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。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。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
15、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。,帽子结构的作用,对mRNA的翻译活性很重要，可使之免受外切核酸酶的降解对mRNA与核糖体的结合包括起始区的辨认也很重要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 plementary to an RNA,synthesized from it by reverse transc
16、ription in vitro体外.,The 3 terminus is polyadenylated,Poly(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,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。与mRNA通过核膜进入细胞质有关与翻译调控有关可利用poly(A)结构在dT分离柱上
17、通过馏分将mRNA与其它RNA分开。,Degradosome降解复合体 is a plex 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 enzymes,Degradation of bacterial mRNA is a two stage process.Endonucleolyti
18、c cleavages proceed 5-3 behind the ribosomes.The released fragments are degraded by exonucleases that move 3-5,Exosome外切体 is a plex of several exonucleases involved in degrading RNA.,Yeast mRNA degradation involves multiple activities,Degradation of yeast mRNA requires deadenylation脱腺苷,decapping去帽,a
19、nd exonucleolysis外切核酸酶降解.,An ARE in a 3 nontranslated region initiates degradation of mRNA.,Sequence elements may destabilize 使不稳定mRNA,ARE:AU-rich sequence,An IRE in a 3 nontranslated region controls mRNA stability.,转铁蛋白mRNA,IRE:Iron regulated element,Surveillance systems check nucleic acids for val
20、idity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 react to damage in the double helix.The mon feature is that the system recognizes an invalid sequence or structure and triggers a resp
21、onse.,Nonsense mutations trigger a surveillance监管 system,Nonsense 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(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
22、。一个携带氨基酸的tRNA叫作氨酰基-tRNA。,Summary,3、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。4、转录机器对于组织和器官不是特定的；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。,5、一条典型的mRNA包括非翻译的5前导区和3尾及编
23、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。6、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，通常少于10个，且每个mRNA仅带有一个编码序列。,7、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。,
