第六章第六章 生物超分子体系生物超分子体系 生物超分子体系生物超分子体系多数是由蛋白质分子或蛋白质分子与多数是由蛋白质分子或蛋白质分子与其他生物大分子，如核酸分子、脂类分子和多糖类分子所其他生物大分子，如核酸分子、脂类分子和多糖类分子所构成的复合体，并表现为超出单一生物大分子各自功能以构成的复合体，并表现为超出单一生物大分子各自功能以外的新功能。外的新功能。 生物超分子体系本身是多组分大分子复生物超分子体系本身是多组分大分子复合体，但不同于简单生物大分子复合物的是合体，但不同于简单生物大分子复合物的是它们呈现出分子机器的功能特征，而不仅仅它们呈现出分子机器的功能特征，而不仅仅是具有结构上的概念。在细胞内发生的一系是具有结构上的概念。在细胞内发生的一系列重要生命活动中列重要生命活动中,都有生物超分子体系起关都有生物超分子体系起关键作用键作用。n n基基因因转转录录中中在在转转录录起起始始阶阶段段出出现的超分子体系；现的超分子体系；n n蛋蛋白白质质翻翻译译合合成成过过程程中中的的核核糖糖体超分子体系；体超分子体系；n n细细胞胞信信号号传传导导过过程程中中的的跨跨膜膜受受体超分子体系；体超分子体系；n n染染色色体体末末端端复复制制过过程程中中涉涉及及到到的端粒酶超分子体系；的端粒酶超分子体系；n n在在蛋蛋白白质质降降解解中中发发挥挥作作用用的的蛋蛋白水解酶超分子体系等。白水解酶超分子体系等。 生命体正是利用生物超分子体系这一生命体正是利用生物超分子体系这一生物活性物质的组织形式，与细胞内的各生物活性物质的组织形式，与细胞内的各种物质协同作用，高效、有序而且可控地种物质协同作用，高效、有序而且可控地完成细胞内的生命活动。完成细胞内的生命活动。 章章 节节 内内 容容n n第一节第一节第一节第一节 生物超分子体系的特征生物超分子体系的特征生物超分子体系的特征生物超分子体系的特征n n第二节第二节第二节第二节 转录阶段的超分子复合体转录阶段的超分子复合体转录阶段的超分子复合体转录阶段的超分子复合体n n第三节第三节第三节第三节 核糖体超分子体系核糖体超分子体系核糖体超分子体系核糖体超分子体系n n第四节第四节第四节第四节 成纤维细胞因子受体成纤维细胞因子受体成纤维细胞因子受体成纤维细胞因子受体- - - -配体超分子配体超分子配体超分子配体超分子 体系体系体系体系 n n第五节第五节第五节第五节 端粒酶超分子体系端粒酶超分子体系端粒酶超分子体系端粒酶超分子体系 第一节第一节 生物超分子体系的特征生物超分子体系的特征 生物超分子体系的生物超分子体系的优越性优越性1.反应的效率化。 2.成分的体系化。 3.控制的反馈性。 4.功能的多样性。 5.高层的识别性。 生物超分子体系的生物超分子体系的分类分类1.1.紧密非共价组合型。紧密非共价组合型。 2.2.离散型。离散型。 3.3.膜结合型。膜结合型。生物超分子体系的人工化研究生物超分子体系的人工化研究n n经过设计，可以运用基因工程手段，经过设计，可以运用基因工程手段，将表达出的单一的单功能蛋白组件集将表达出的单一的单功能蛋白组件集成化，形成新的生物超分子体系。成化，形成新的生物超分子体系。n n利用分子识别与催化功能，以及生物利用分子识别与催化功能，以及生物超分子自组装功能，发展分子传感器超分子自组装功能，发展分子传感器技术，以及人工膜系统，技术，以及人工膜系统，n n利用生物芯片组装发展生物计算机。利用生物芯片组装发展生物计算机。第二节第二节 转录阶段的超分子转录阶段的超分子复合体复合体n n在基因的转录起始阶段，多种因子在启动在基因的转录起始阶段，多种因子在启动区上聚集与相继解离，构成了转录起始超区上聚集与相继解离，构成了转录起始超分子体系，形成一部转录起始的机器。分子体系，形成一部转录起始的机器。n n精细的、高层次的识别反应性，以及集成精细的、高层次的识别反应性，以及集成化的组装，基因转录体系中各种转录因子化的组装，基因转录体系中各种转录因子之间，以及各种转录因子与特定碱基序列之间，以及各种转录因子与特定碱基序列之间的精确识别能力，共同决定了转录起之间的精确识别能力，共同决定了转录起始超分子体系内极高的反应能力。始超分子体系内极高的反应能力。 RNARNA聚合酶转录超分子体系的组成聚合酶转录超分子体系的组成n nDNA 启动子、启动子、DNA调节元件调节元件n n蛋白因子蛋白因子 1.RNA1.RNA聚合酶和基本转录因子聚合酶和基本转录因子( (GTFsGTFs) )。 2.2.调节蛋白调节蛋白, ,如激活因子和抑制因子。如激活因子和抑制因子。 3.3.协调因子。协调因子。原核细胞的转录原核细胞的转录 Figure. E. coli RNA polymerase The complete enzyme consists of five subunits: two , one , one , and one . The subunit is relatively weakly bound and can be dissociated from the other four subunits, which constitute the core polymerase. Figure. Sequences of E. coli promoters E. coli promoters are characterized by two sets of sequences located 10 and 35 base pairs upstream of the transcription start site (+1). The consensus sequences shown correspond to the bases most frequently found in different promoters. Figure. Transcription by E. coli RNA polymerase The polymerase initially binds nonspecifically to DNA and migrates along the molecule until the subunit binds to the -35 and -10 promoter elements, forming a closed-promoter complex. The polymerase then unwinds DNA around the initiation site, and transcription is initiated by the polymerization of free NTPs. The subunit then dissociates from the core polymerase, which migrates along the DNA and elongates the growing RNA chain. Figure. Transcription termination The termination of transcription is signaled by a GC-rich inverted repeat followed by four A residues. The inverted repeat forms a stable stem-loop structure in the RNA, causing the RNA to dissociate from the DNA template. 真核细胞的转录真核细胞的转录Figure. Formation of a polymerase II transcription complex Many polymerase II promoters have a TATA box (consensus sequence TATAA) 25 to 30 nucleotides upstream of the transcription start site. This sequence is recognized by transcription factor TFIID, which consists of the TATA-binding protein (TBP) and TBP-associated factors (TAFs). TFIIB(B) then binds to TBP, followed by binding of the polymerase in association with TFIIF(F). Finally, TFIIE(E) and TFIIH(H) associate with the complex.Figure. RNA polymerase II holoenzyme The holoenzyme consists of a preformed complex of RNA polymerase II, the general transcription factors TFIIB, TFIIE, TFIIF, and TFIIH, and several other proteins that activate transcription. This complex can be recruited directly to a promoter via interaction with TFIID (TBP + TAFs). 20002000年已获得年已获得RNARNA聚合酶聚合酶IIII和和RNARNA聚合酶聚合酶IIII结合一个转结合一个转录因子的晶体模型。录因子的晶体模型。 真核转录中的基本转录因子真核转录中的基本转录因子(GTFs) n nTFIIAn nTFIIBn nTFIIDn nTFIIEn nTFIIFn nTFIIH n n调节蛋白调节蛋白(激活因子和抑制因子激活因子和抑制因子) n n协调因子协调因子n n中介因子中介因子 n n TFIID is itself composed of multiple subunits, TFIID is itself composed of multiple subunits, including the including the TATA-binding proteinTATA-binding protein (TBP), which (TBP), which binds specifically to the TATAA consensus binds specifically to the TATAA consensus sequence, and 10-12 other polypeptides, called sequence, and 10-12 other polypeptides, called TBP-associated factors TBP-associated factors ( (TAFsTAFs). ). TBP then binds a TBP then binds a second general transcription factor (TFIIB) second general transcription factor (TFIIB) forming a TBP-TFIIB complex at the promoter. forming a TBP-TFIIB complex at the promoter. TFIIB in turn serves as a bridge to RNA TFIIB in turn serves as a bridge to RNA polymerase, which binds to the TBP-TFIIB polymerase, which binds to the TBP-TFIIB complex in association with a third factor, TFIIF.complex in association with a third factor, TFIIF.n nFollowing recruitment of RNA polymerase II to the promoter, Following recruitment of RNA polymerase II to the promoter, the binding of two additional factors (TFIIE and TFIIH) is the binding of two additional factors (TFIIE and TFIIH) is required for initiation of transcription. required for initiation of transcription. n nTFIIH is a TFIIH is a multisubunitmultisubunit factor that appears to play at least factor that appears to play at least two important roles. First, two subunits of TFIIH are two important roles. First, two subunits of TFIIH are helicaseshelicases, which may unwind DNA around the initiation site. , which may unwind DNA around the initiation site. (These subunits of TFIIH are also required for nucleotide (These subunits of TFIIH are also required for nucleotide excision repair. Another subunit of TFIIH is a protein excision repair. Another subunit of TFIIH is a protein kinasekinase that that phosphorylatesphosphorylates repeated sequences present in the C- repeated sequences present in the C-terminal domain of the largest subunit of RNA polymerase II. terminal domain of the largest subunit of RNA polymerase II. PhosphorylationPhosphorylation of these sequences is thought to release the of these sequences is thought to release the polymerase from its association with the initiation complex, polymerase from its association with the initiation complex, allowing it to proceed along the template as it elongates the allowing it to proceed along the template as it elongates the growing RNA chain.growing RNA chain.Figure. Action of enhancers Without an enhancer, the gene is transcribed at a low basal level (A). Addition of an enhancer, Efor example, the SV40 72-base-pair repeatsstimulates transcription. The enhancer is active not only when placed just upstream of the promoter (B), but also when inserted up to several kilobases either upstream or downstream from the transcription start site (C and D). In addition, enhancers are active in either the forward or backward orientation (E). Figure. DNA looping Transcription factors bound at distant enhancers are able to interact with general transcription factors at the promoter because the intervening DNA can form loops. There is therefore no fundamental difference between the action of transcription factors bound to DNA just upstream of the promoter and to distant enhancers. Figure. Structure of transcriptional activators Transcriptional activators consist of two independent domains. The DNA-binding domain recognizes a specific DNA sequence, and the activation domain interacts with other components of the transcriptional machinery. Figure. Synergistic action of transcriptional activators Different transcriptional activators can interact with the general transcription factor TFIID by binding to different TAFs Figure. Action of eukaryotic repressors (A) Some repressors block the binding of activators to regulatory sequences. (B) Other repressors have active repression domains that inhibit transcription by interactions with general transcription factors. Figure. Histone acetylation (A) The core histones have histone-fold domains, which interact with other histones and with DNA in the nucleosome, and N-terminal tails, which extend outside of the nucleosome. The N-terminal tails of the core histones (e.g., H3) are modified by the addition of acetyl groups (Ac) to the side chains of specific lysine residues. (B) Transcriptional activators and repressors are associated with coactivators and corepressors, which have histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities, respectively. Histone acetylation is characteristic of actively transcribed chromatin and may weaken the binding of histones to DNA or alter their interactions with other proteins. 基基因因表表达达的的激激活活 SummarySummary思考题思考题n n生物超分子体系n nGTFs n n生物超分子体系的功能和特征如何？n n谈谈转录起始阶段的超分子复合体。第三节第三节 核糖体超分子体系核糖体超分子体系蛋白质分子翻译中的主要元件蛋白质分子翻译中的主要元件n nmRNAn ntRNAn n核糖体n n其他翻译相关蛋白Figure. Prokaryotic and eukaryotic mRNAs Both prokaryotic and eukaryotic mRNAs contain untranslated regions (UTRs) at their 5 and 3 ends. Eukaryotic mRNAs also contain 5 7-methylguanosine (m7G) caps and 3 poly-A tails. Prokaryotic mRNAs are frequently polycistronic: They encode multiple proteins, each of which is translated from an independent start site. Eukaryotic mRNAs are usually monocistronic, encoding only a single protein. Figure. Structure of tRNAs The structure of yeast phenylalanyl tRNA is illustrated in open “cloverleaf” form (A) to show complementary base pairing. Modified bases are indicated as mG, methylguanosine; mC, methylcytosine; DHU, dihydrouridine; T, ribothymidine; Y, a modified purine (usually adenosine); and y, pseudouridine. The folded form of the molecule is shown in (B) and a space-filling model in (C). (C, courtesy of Dan Richardson.)Figure. Attachment of amino acids to tRNAs In the first reaction step, the amino acid is joined to AMP, forming an aminoacyl AMP intermediate. In the second step, the amino acid is transferred to the 3 CCA terminus of the acceptor tRNA and AMP is released. Both steps of the reaction are catalyzed by aminoacyl tRNA synthetases. 核糖体是一个非常巨大的核糖核蛋白颗核糖体是一个非常巨大的核糖核蛋白颗粒，作为蛋白质合成的场所，在翻译过程粒，作为蛋白质合成的场所，在翻译过程中，核糖体与众多翻译辅助因子，以及各中，核糖体与众多翻译辅助因子，以及各种种RNA分子自动高度有序地组装成一个超分子自动高度有序地组装成一个超分子体系，这种组装赋予了超分子体系基分子体系，这种组装赋予了超分子体系基元单体所不具有的特异的化学、物理、生元单体所不具有的特异的化学、物理、生物或智能的特性，使各基元单体在同一空物或智能的特性，使各基元单体在同一空间内共同执行翻译功能，让肽链的合成有间内共同执行翻译功能，让肽链的合成有条不紊的进行。条不紊的进行。 核糖体的自我组装核糖体的自我组装 n n核糖体是一种自组装颗粒，可以由专一性的核糖体是一种自组装颗粒，可以由专一性的核糖体是一种自组装颗粒，可以由专一性的核糖体是一种自组装颗粒，可以由专一性的RNARNA和蛋白分子结合形成具有活性的超分子体系。在和蛋白分子结合形成具有活性的超分子体系。在和蛋白分子结合形成具有活性的超分子体系。在和蛋白分子结合形成具有活性的超分子体系。在自我组装的过程中，其需要的全部信息来源都在自我组装的过程中，其需要的全部信息来源都在自我组装的过程中，其需要的全部信息来源都在自我组装的过程中，其需要的全部信息来源都在亚基结构里，其蛋白质和亚基结构里，其蛋白质和亚基结构里，其蛋白质和亚基结构里，其蛋白质和rRNArRNA都带有规定组装过都带有规定组装过都带有规定组装过都带有规定组装过程的全部信息。自我组装的驱动力包括疏水性作程的全部信息。自我组装的驱动力包括疏水性作程的全部信息。自我组装的驱动力包括疏水性作程的全部信息。自我组装的驱动力包括疏水性作用力，氢键和离子相互作用，及碱基堆叠之间的用力，氢键和离子相互作用，及碱基堆叠之间的用力，氢键和离子相互作用，及碱基堆叠之间的用力，氢键和离子相互作用，及碱基堆叠之间的相互作用等。相互作用等。相互作用等。相互作用等。 n n另外，这个组装有一定的顺序，即某蛋白的加入另外，这个组装有一定的顺序，即某蛋白的加入另外，这个组装有一定的顺序，即某蛋白的加入另外，这个组装有一定的顺序，即某蛋白的加入优先与其他的蛋白质。而且各组分的加入是有协优先与其他的蛋白质。而且各组分的加入是有协优先与其他的蛋白质。而且各组分的加入是有协优先与其他的蛋白质。而且各组分的加入是有协同作用的，一种组分的加入加强了下一种组分的同作用的，一种组分的加入加强了下一种组分的同作用的，一种组分的加入加强了下一种组分的同作用的，一种组分的加入加强了下一种组分的加入。加入。加入。加入。 原核生物核糖体的结构原核生物核糖体的结构n nsmall subunit (designated 30S) of E. coli ribosomes consists of the 16S rRNA and 21 proteins; the large subunit (50S) is composed of the 23S and 5S rRNAs and 34 proteins. Each ribosome contains one copy of the rRNAs and one copy of each of the ribosomal proteins, with one exception: One protein of the 50S subunit is present in four copies. （A）16S rRNA的结构域的结构域 （B）30S subunit的四级结构的四级结构 A.aeolicus 16S rRNA的二级结构，图示核糖体蛋白与的二级结构，图示核糖体蛋白与16S rRNA（灰色骨架）的联系（灰色骨架）的联系 23S rRNA的二维图的二维图 70S核糖体的连接区域核糖体的连接区域(a)从从30S亚基角度看连接区域亚基角度看连接区域(b) 从从50S亚基角度看连接区域亚基角度看连接区域真核生物核糖体的结构真核生物核糖体的结构n nThe subunits of eukaryotic ribosomes are larger and contain more proteins than their prokaryotic counterparts have. The small subunit (40S) of eukaryotic ribosomes is composed of the 18S rRNA and approximately 30 proteins; the large subunit (60S) contains the 28S, 5.8S, and 5S rRNAs and about 45 proteins. n n组装后的真核生物核糖体与原核生物核糖体具有组装后的真核生物核糖体与原核生物核糖体具有组装后的真核生物核糖体与原核生物核糖体具有组装后的真核生物核糖体与原核生物核糖体具有相似的结构和功能。但它们在细节上有很大不同，相似的结构和功能。但它们在细节上有很大不同，相似的结构和功能。但它们在细节上有很大不同，相似的结构和功能。但它们在细节上有很大不同，包括质量和亚基的成分。包括质量和亚基的成分。包括质量和亚基的成分。包括质量和亚基的成分。n n小亚基小亚基小亚基小亚基40S40S含有含有含有含有3333个特异肽链和个特异肽链和个特异肽链和个特异肽链和18S 18S rRNArRNA。大亚。大亚。大亚。大亚基有基有基有基有4949个不同的肽链和个不同的肽链和个不同的肽链和个不同的肽链和3 3个个个个rRNArRNA（28S28S，5.8S, 5.8S, 5S5S）。）。）。）。n n28S28S和和和和18S 18S rRNArRNA的二级结构与原核的的二级结构与原核的的二级结构与原核的的二级结构与原核的16S16S，23S 23S rRNArRNA相似，相似，相似，相似，5.8S 5.8S rRNArRNA与与与与28S 28S rRNArRNA形成碱基配形成碱基配形成碱基配形成碱基配对，它们与原核对，它们与原核对，它们与原核对，它们与原核23S 23S rRNArRNA的的的的55末端序列具有同源末端序列具有同源末端序列具有同源末端序列具有同源性。性。性。性。 图图 真核与原核生物核糖体结构真核与原核生物核糖体结构(a,b,c)真核生物真核生物80S核糖体核糖体；(d,e,f)为原核生物为原核生物70S核糖体；核糖体；(g,h)为为80S核糖体大大小亚基；核糖体大大小亚基；(i,j)为为70S核糖体大小亚基。核糖体大小亚基。Figure. Ribosome structure (D) Model of ribosome structure. (E) Components of prokaryotic and eukaryotic ribosomes. Intact prokaryotic and eukaryotic ribosomes are designated 70S and 80S, respectively, on the basis of their sedimentation rates in ultracentrifugation. They consist of large and small subunits, which contain both ribosomal proteins and rRNAs. 真核生物线粒体的核糖体的结构真核生物线粒体的核糖体的结构 n n线粒体涉及核糖体的基因组成分显著减少，核糖线粒体涉及核糖体的基因组成分显著减少，核糖线粒体涉及核糖体的基因组成分显著减少，核糖线粒体涉及核糖体的基因组成分显著减少，核糖体的体的体的体的RNARNA成份在长度方面减少，蛋白质成分增加。成份在长度方面减少，蛋白质成分增加。成份在长度方面减少，蛋白质成分增加。成份在长度方面减少，蛋白质成分增加。n n图中模型显示了图中模型显示了图中模型显示了图中模型显示了93%93%的的的的RNARNA成分及大亚基的成分及大亚基的成分及大亚基的成分及大亚基的1616个个个个核糖体蛋白，虽然核糖体蛋白，虽然核糖体蛋白，虽然核糖体蛋白，虽然rRNArRNA变小了，但变小了，但变小了，但变小了，但rRNArRNA的结构的结构的结构的结构域仍直接对蛋白质的合成起作用。域仍直接对蛋白质的合成起作用。域仍直接对蛋白质的合成起作用。域仍直接对蛋白质的合成起作用。n n另外另外另外另外, , rRNArRNA变小之后限制了变小之后限制了变小之后限制了变小之后限制了tRNAtRNA与核糖体与核糖体与核糖体与核糖体EE位位位位点的结合，且与点的结合，且与点的结合，且与点的结合，且与tRNAtRNA的的的的D-D-环和环和环和环和T T环的变小有关。环的变小有关。环的变小有关。环的变小有关。真真核核生生物物线线粒粒体体的的核核糖糖体体 蛋白质翻译过程中的超分子体系蛋白质翻译过程中的超分子体系 n n蛋白质翻译过程，涉及蛋白质翻译过程，涉及蛋白质翻译过程，涉及蛋白质翻译过程，涉及mRNAmRNA，tRNAtRNA，rRNArRNA和和和和2020种型的氨基酸，几种核苷酸（，种型的氨基酸，几种核苷酸（，种型的氨基酸，几种核苷酸（，种型的氨基酸，几种核苷酸（，）以及一系列酶，各种蛋白辅助因子。大约有）以及一系列酶，各种蛋白辅助因子。大约有）以及一系列酶，各种蛋白辅助因子。大约有）以及一系列酶，各种蛋白辅助因子。大约有将近种细胞成分参加了蛋白质的生物合成。将近种细胞成分参加了蛋白质的生物合成。将近种细胞成分参加了蛋白质的生物合成。将近种细胞成分参加了蛋白质的生物合成。n n合成大致分为三个阶段。）肽链合成的起始阶合成大致分为三个阶段。）肽链合成的起始阶合成大致分为三个阶段。）肽链合成的起始阶合成大致分为三个阶段。）肽链合成的起始阶段，）肽链的延伸，）肽链合成的终止与释段，）肽链的延伸，）肽链合成的终止与释段，）肽链的延伸，）肽链合成的终止与释段，）肽链的延伸，）肽链合成的终止与释放。蛋白质合成体系可以说是高度组织化的超分放。蛋白质合成体系可以说是高度组织化的超分放。蛋白质合成体系可以说是高度组织化的超分放。蛋白质合成体系可以说是高度组织化的超分子体系，保证了蛋白质生物合成的正确性和高效子体系，保证了蛋白质生物合成的正确性和高效子体系，保证了蛋白质生物合成的正确性和高效子体系，保证了蛋白质生物合成的正确性和高效性。性。性。性。n nTranslation is generally divided into three stages: Translation is generally divided into three stages: initiation, elongation, and termination. initiation, elongation, and termination. n nIn both prokaryotes and eukaryotes the first step of In both prokaryotes and eukaryotes the first step of the initiation stage is the binding of a specific initiator the initiation stage is the binding of a specific initiator methionylmethionyl tRNAtRNA and the mRNA to the small and the mRNA to the small ribosomal subunit. The large ribosomal subunit then ribosomal subunit. The large ribosomal subunit then joins the complex, forming a functional ribosome on joins the complex, forming a functional ribosome on which elongation of the polypeptide chain proceeds. which elongation of the polypeptide chain proceeds. A number of specific A number of specific nonribosomalnonribosomal proteins are also proteins are also required for the various stages of the translation required for the various stages of the translation process process Figure. Overview of translation 肽链合成的起始阶段的超分子体系肽链合成的起始阶段的超分子体系 n nThe first translation step in bacteria is the binding of three The first translation step in bacteria is the binding of three initiation factorsinitiation factors (IF-1, IF-2, and IF-3) to the 30S ribosomal (IF-1, IF-2, and IF-3) to the 30S ribosomal subunit. The mRNA and initiator subunit. The mRNA and initiator N N- -formylmethionylformylmethionyl tRNAtRNA then join the complex, with IF-2 (which is bound to GTP) then join the complex, with IF-2 (which is bound to GTP) specifically recognizing the initiator specifically recognizing the initiator tRNAtRNA. IF-3 is then . IF-3 is then released, allowing a 50S ribosomal subunit to associate with released, allowing a 50S ribosomal subunit to associate with the complex. This association triggers the hydrolysis of GTP the complex. This association triggers the hydrolysis of GTP bound to IF-2, which leads to the release of IF-1 and IF-2 bound to IF-2, which leads to the release of IF-1 and IF-2 (bound to GDP). The result is the formation of a 70S initiation (bound to GDP). The result is the formation of a 70S initiation complex (with mRNA and initiator complex (with mRNA and initiator tRNAtRNA bound to the bound to the ribosome) that is ready to begin peptide bond formation during ribosome) that is ready to begin peptide bond formation during the elongation stage of translation. the elongation stage of translation. Figure. Initiation of translation in bacteria Three initiation factors (IF-1, IF-2, and IF-3) first bind to the 30S ribosomal subunit. This step is followed by binding of the mRNA and the initiator N-formylmethionyl (fMet) tRNA, which is recognized by IF-2 bound to GTP. IF-3 is then released, and a 50S subunit binds to the complex, triggering the hydrolysis of bound GTP, followed by the release of IF-1 and IF-2 bound to GDP. n nInitiation in eukaryotes is more complicated and requires Initiation in eukaryotes is more complicated and requires at least ten proteins (each consisting of multiple at least ten proteins (each consisting of multiple polypeptide chains), which are designated polypeptide chains), which are designated eIFseIFs ( (e eukaryotic ukaryotic i initiation nitiation f factors). actors). n nThe factors eIF-1, eIF-1A, and eIF-3 bind to the 40S The factors eIF-1, eIF-1A, and eIF-3 bind to the 40S ribosomal subunit, and eIF-2 (in a complex with GTP) ribosomal subunit, and eIF-2 (in a complex with GTP) associates with the initiator associates with the initiator methionylmethionyl tRNAtRNA. The mRNA . The mRNA is recognized and brought to the ribosome by the eIF-4 is recognized and brought to the ribosome by the eIF-4 group of factors. The 5 cap of the mRNA is recognized group of factors. The 5 cap of the mRNA is recognized by eIF-4E. by eIF-4E. n nAnother factor, eIF-4G, binds to both eIF-4E and to a Another factor, eIF-4G, binds to both eIF-4E and to a protein (poly-A binding protein or PABP) associated with protein (poly-A binding protein or PABP) associated with the poly-A tail at the 3 end of the mRNA. the poly-A tail at the 3 end of the mRNA. n nEukaryotic initiation factors thus recognize both the 5 and 3 Eukaryotic initiation factors thus recognize both the 5 and 3 ends of mRNAs, accounting for the stimulatory effect of ends of mRNAs, accounting for the stimulatory effect of polyadenylationpolyadenylation on translation. The initiation factors eIF-4E on translation. The initiation factors eIF-4E and eIF-4G, in association with eIF-4A and eIF-4B, then bring and eIF-4G, in association with eIF-4A and eIF-4B, then bring the mRNA to the 40S ribosomal subunit, with eIF-4G the mRNA to the 40S ribosomal subunit, with eIF-4G interacting with eIF-3. The 40S ribosomal subunit, in interacting with eIF-3. The 40S ribosomal subunit, in association with the bound association with the bound methionylmethionyl tRNAtRNA and and eIFseIFs, then , then scans the mRNA to identify the AUG initiation scans the mRNA to identify the AUG initiation codoncodon. When . When the AUG the AUG codoncodon is reached, eIF-5 triggers the hydrolysis of is reached, eIF-5 triggers the hydrolysis of GTP bound to eIF-2. Initiation factors (including eIF-2 bound GTP bound to eIF-2. Initiation factors (including eIF-2 bound to GDP) are then released, and a 60S subunit binds to the 40S to GDP) are then released, and a 60S subunit binds to the 40S subunit to form the 80S initiation complex of eukaryotic cells.subunit to form the 80S initiation complex of eukaryotic cells.Figure. Initiation of translation in eukaryotic cells Initiation factors eIF-3, eIF-1, and eIF-1A bind to the 40S ribosomal subunit. The initiator methionyl tRNA is brought to the ribosome by eIF-2 (complexed to GTP), and the mRNA by eIF-4E (which binds to the 5 cap), eIF-4G (which binds to both eIF-4E at the 5 cap and PABP at the 3 poly-A tail), eIF-4A, and eIF-4B. The ribosome then scans down the mRNA to identify the first AUG initiation codon. Scanning requires energy and is accompanied by ATP hydrolysis. When the initiating AUG is identified, eIF-5 triggers the hydrolysis of GTP bound to eIF-2, followed by the release of eIF-2 (complexed to GDP) and other initiation factors. The 60S ribosomal subunit then joins the 40S complex. 肽链合成的延伸阶段的超分子复合体肽链合成的延伸阶段的超分子复合体 n n肽链延伸由许多循环组成，每加入一个氨基酸就是一次循环，每次循环包括三步：1）AA-tRNA与核糖体的结合，2）转肽，3）移位。n n在每一循环步骤中，核糖体与各种延伸因子形成超分子体系，提供肽链延伸的空间和能量。n n另外，最近还发现RRF（ribosome recycling factor），它与延伸因子EF-G有相互依存的作用。 Figure. Elongation stage of translation The ribosome has three tRNA-binding sites, designated P (peptidyl), A (aminoacyl), and E (exit). The initiating N-formylmethionyl tRNA is positioned in the P site, leaving an empty A site. The second aminoacyl tRNA (e.g., alanyl tRNA) is then brought to the A site by EF-Tu (complexed with GTP). Following GTP hydrolysis, EF-Tu (complexed with GDP) leaves the ribosome, with alanyl tRNA inserted into the A site. A peptide bond is then formed, resulting in the transfer of methionine to the aminoacyl tRNA at the A site. The ribosome then moves three nucleotides along the mRNA. This movement translocates the peptidyl (Met-Ala) tRNA to the P site and the uncharged tRNA to the E site, leaving an empty A site ready for addition of the next amino acid. Translocation is mediated by EF-G, coupled to GTP hydrolysis. The process, illustrated here for prokaryotic cells, is very similar in eukaryotes. 图 RRF与70Sribosome作用的结构模型A）RRF与EF-GdomainII在70S核糖体中的定位；B）EF-G结合到核糖体上后会促使RRF-e2的定位；C）模型显示RRF的结构域II和链合区与EF-G的结构域II，III之间的相互作用；D）70S-RRF中EF-G(green)的定位,显示了EF-G的domainsIV和V与RRF-e2的domainII之间的作用 图图 延伸过程中的整个核糖体结构，还显示有延伸过程中的整个核糖体结构，还显示有A-和和 P-sites的的tRNAs结合位点结合位点肽链合成的终止肽链合成的终止 n n原核细胞的肽链合成的终止需要有原核细胞的肽链合成的终止需要有RF-1/RF2RF-1/RF2、RF-RF-3 3的参与；的参与；RF-1RF-1识别识别UAAUAA和和UAGUAG，RF2RF2识别识别UAAUAA和和UGAUGA，RF-3RF-3则与则与GTPGTP结合并与结合并与RF-1/RF-2RF-1/RF-2、终止、终止密码子形成复合物，并改变肽酰转移酶的特异性，密码子形成复合物，并改变肽酰转移酶的特异性，使肽酰转移酶水解使肽酰转移酶水解P P位点上的多肽链和位点上的多肽链和tRNAtRNA之间之间的链，随后新生肽链和的链，随后新生肽链和tRNAtRNA与核糖体解离与核糖体解离, , 肽链肽链合成终止。合成终止。n n真核细胞肽链合成的终止只有一种真核细胞肽链合成的终止只有一种RFRF（表示为（表示为eRFeRF），它代替了原核的），它代替了原核的RF-1RF-1、RF-2RF-2和和RF-3RF-3。Figure. Termination of translation A termination codon (e.g., UAA) at the A site is recognized by a release factor rather than by a tRNA. The result is the release of the completed polypeptide chain, followed by the dissociation of tRNA and mRNA from the ribosome. 图图 (a) T.maritima的的RF1 X-衍射结构，衍射结构，(b) E.coli的的RF2 X-衍射结构衍射结构Summary思考题思考题n n核糖体超分子体系是如何组装的？n n从理论上讲，应该如何人工设计并组装一个生物超分子体系？