分子生物學(xué)：第六章 生物超分子體系

1、第六章 生物超分子體系 生物超分子體系多數(shù)是由蛋白質(zhì)分子或蛋白質(zhì)分子與其他生物大分子，如核酸分子、脂類分子和多糖類分子所構(gòu)成的復(fù)合體，并表現(xiàn)為超出單一生物大分子各自功能以外的新功能。 生物超分子體系本身是多組分大分子復(fù)合體，但不同于簡單生物大分子復(fù)合物的是它們呈現(xiàn)出分子機(jī)器的功能特征，而不僅僅是具有結(jié)構(gòu)上的概念。在細(xì)胞內(nèi)發(fā)生的一系列重要生命活動(dòng)中,都有生物超分子體系起關(guān)鍵作用?；蜣D(zhuǎn)錄中在轉(zhuǎn)錄起始階段出現(xiàn)的超分子體系；蛋白質(zhì)翻譯合成過程中的核糖體超分子體系；細(xì)胞信號傳導(dǎo)過程中的跨膜受體超分子體系；染色體末端復(fù)制過程中涉及到的端粒酶超分子體系；在蛋白質(zhì)降解中發(fā)揮作用的蛋白水解酶超分子體系等。 生

2、命體正是利用生物超分子體系這一生物活性物質(zhì)的組織形式，與細(xì)胞內(nèi)的各種物質(zhì)協(xié)同作用，高效、有序而且可控地完成細(xì)胞內(nèi)的生命活動(dòng)。 章 節(jié) 內(nèi) 容第一節(jié) 生物超分子體系的特征第二節(jié) 轉(zhuǎn)錄階段的超分子復(fù)合體第三節(jié) 核糖體超分子體系第四節(jié) 成纖維細(xì)胞因子受體-配體超分子 體系 第五節(jié) 端粒酶超分子體系 第一節(jié) 生物超分子體系的特征 生物超分子體系的優(yōu)越性1.反應(yīng)的效率化。 2.成分的體系化。 3.控制的反饋性。 4.功能的多樣性。 5.高層的識別性。 生物超分子體系的分類1.緊密非共價(jià)組合型。 2.離散型。 3.膜結(jié)合型。生物超分子體系的人工化研究經(jīng)過設(shè)計(jì)，可以運(yùn)用基因工程手段，將表達(dá)出的單一的單功能蛋

3、白組件集成化，形成新的生物超分子體系。利用分子識別與催化功能，以及生物超分子自組裝功能，發(fā)展分子傳感器技術(shù)，以及人工膜系統(tǒng)，利用生物芯片組裝發(fā)展生物計(jì)算機(jī)。第二節(jié) 轉(zhuǎn)錄階段的超分子復(fù)合體在基因的轉(zhuǎn)錄起始階段，多種因子在啟動(dòng)區(qū)上聚集與相繼解離，構(gòu)成了轉(zhuǎn)錄起始超分子體系，形成一部轉(zhuǎn)錄起始的機(jī)器。精細(xì)的、高層次的識別反應(yīng)性，以及集成化的組裝，基因轉(zhuǎn)錄體系中各種轉(zhuǎn)錄因子之間，以及各種轉(zhuǎn)錄因子與特定堿基序列之間的精確識別能力，共同決定了轉(zhuǎn)錄起始超分子體系內(nèi)極高的反應(yīng)能力。 RNA聚合酶轉(zhuǎn)錄超分子體系的組成DNA 啟動(dòng)子、DNA調(diào)節(jié)元件蛋白因子 1.RNA聚合酶和基本轉(zhuǎn)錄因子(GTFs)。 2.調(diào)節(jié)蛋白,

4、如激活因子和抑制因子。 3.協(xié)調(diào)因子。原核細(xì)胞的轉(zhuǎn)錄 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 promoter

5、s 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 polymeras

6、e 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

7、 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

8、structure in the RNA, causing the RNA to dissociate from the DNA template. 真核細(xì)胞的轉(zhuǎn)錄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 recogni

9、zed 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 h

10、oloenzyme 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). 2000年

11、已獲得RNA聚合酶II和RNA聚合酶II結(jié)合一個(gè)轉(zhuǎn)錄因子的晶體模型。 真核轉(zhuǎn)錄中的基本轉(zhuǎn)錄因子(GTFs) TFIIATFIIBTFIIDTFIIETFIIFTFIIH 調(diào)節(jié)蛋白(激活因子和抑制因子) 協(xié)調(diào)因子中介因子 TFIID is itself composed of multiple subunits, including the TATA-binding protein (TBP), which binds specifically to the TATAA consensus sequence, and 10-12 other polypeptides, called TBP-as

12、sociated factors (TAFs). TBP then binds a second general transcription factor (TFIIB) forming a TBP-TFIIB complex at the promoter. TFIIB in turn serves as a bridge to RNA polymerase, which binds to the TBP-TFIIB complex in association with a third factor, TFIIF.Following recruitment of RNA polymeras

13、e II to the promoter, the binding of two additional factors (TFIIE and TFIIH) is required for initiation of transcription. TFIIH is a multisubunit factor that appears to play at least two important roles. First, two subunits of TFIIH are helicases, which may unwind DNA around the initiation site. (T

14、hese subunits of TFIIH are also required for nucleotide excision repair. Another subunit of TFIIH is a protein kinase that phosphorylates repeated sequences present in the C-terminal domain of the largest subunit of RNA polymerase II. Phosphorylation of these sequences is thought to release the poly

15、merase from its association with the initiation complex, allowing it to proceed along the template as it elongates the 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 rep

16、eatsstimulates 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 o

17、rientation (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 D

18、NA 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 transc

19、riptional 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 regu

20、latory 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-t

21、erminal 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 corepresso

22、rs, 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. 基因表達(dá)的激活 SummarySummary思考題生物超分

23、子體系GTFs 生物超分子體系的功能和特征如何？談?wù)勣D(zhuǎn)錄起始階段的超分子復(fù)合體。第三節(jié) 核糖體超分子體系蛋白質(zhì)分子翻譯中的主要元件mRNAtRNA核糖體其他翻譯相關(guān)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 tail

24、s. 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

25、 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 spa

26、ce-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

27、 is released. Both steps of the reaction are catalyzed by aminoacyl tRNA synthetases. 核糖體是一個(gè)非常巨大的核糖核蛋白顆粒，作為蛋白質(zhì)合成的場所，在翻譯過程中，核糖體與眾多翻譯輔助因子，以及各種RNA分子自動(dòng)高度有序地組裝成一個(gè)超分子體系，這種組裝賦予了超分子體系基元單體所不具有的特異的化學(xué)、物理、生物或智能的特性，使各基元單體在同一空間內(nèi)共同執(zhí)行翻譯功能，讓肽鏈的合成有條不紊的進(jìn)行。 核糖體的自我組裝 核糖體是一種自組裝顆粒，可以由專一性的RNA和蛋白分子結(jié)合形成具有活性的超分子體系。在自我組裝的過程中，其

28、需要的全部信息來源都在亞基結(jié)構(gòu)里，其蛋白質(zhì)和rRNA都帶有規(guī)定組裝過程的全部信息。自我組裝的驅(qū)動(dòng)力包括疏水性作用力，氫鍵和離子相互作用，及堿基堆疊之間的相互作用等。 另外，這個(gè)組裝有一定的順序，即某蛋白的加入優(yōu)先與其他的蛋白質(zhì)。而且各組分的加入是有協(xié)同作用的，一種組分的加入加強(qiáng)了下一種組分的加入。 原核生物核糖體的結(jié)構(gòu)small subunit (designated 30S) of E. coli ribosomes consists of the 16S rRNA and 21 proteins; the large subunit (50S) is composed of the 23S

29、 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的結(jié)構(gòu)域 （B）30S subunit的四級結(jié)構(gòu) A.aeolicus 16S rRNA的二級結(jié)構(gòu)，圖示核糖體蛋白與16S rRNA（灰色骨架）的聯(lián)系 23S rRNA的二維

30、圖 70S核糖體的連接區(qū)域(a)從30S亞基角度看連接區(qū)域(b) 從50S亞基角度看連接區(qū)域真核生物核糖體的結(jié)構(gòu)The 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 (60

31、S) contains the 28S, 5.8S, and 5S rRNAs and about 45 proteins. 組裝后的真核生物核糖體與原核生物核糖體具有相似的結(jié)構(gòu)和功能。但它們在細(xì)節(jié)上有很大不同，包括質(zhì)量和亞基的成分。小亞基40S含有33個(gè)特異肽鏈和18S rRNA。大亞基有49個(gè)不同的肽鏈和3個(gè)rRNA（28S，5.8S, 5S）。28S和18S rRNA的二級結(jié)構(gòu)與原核的16S，23S rRNA相似，5.8S rRNA與28S rRNA形成堿基配對，它們與原核23S rRNA的5末端序列具有同源性。 圖 真核與原核生物核糖體結(jié)構(gòu)(a,b,c)真核生物80S核糖體；(d,e,

32、f)為原核生物70S核糖體；(g,h)為80S核糖體大大小亞基；(i,j)為70S核糖體大小亞基。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 ultr

33、acentrifugation. They consist of large and small subunits, which contain both ribosomal proteins and rRNAs. 真核生物線粒體的核糖體的結(jié)構(gòu) 線粒體涉及核糖體的基因組成分顯著減少，核糖體的RNA成份在長度方面減少，蛋白質(zhì)成分增加。圖中模型顯示了93%的RNA成分及大亞基的16個(gè)核糖體蛋白，雖然rRNA變小了，但rRNA的結(jié)構(gòu)域仍直接對蛋白質(zhì)的合成起作用。另外, rRNA變小之后限制了tRNA與核糖體E位點(diǎn)的結(jié)合，且與tRNA的D-環(huán)和T環(huán)的變小有關(guān)。真核生物線粒體的核糖體 蛋白質(zhì)翻譯過程中的

34、超分子體系 蛋白質(zhì)翻譯過程，涉及mRNA，tRNA，rRNA和20種型的氨基酸，幾種核苷酸（，）以及一系列酶，各種蛋白輔助因子。大約有將近種細(xì)胞成分參加了蛋白質(zhì)的生物合成。合成大致分為三個(gè)階段。）肽鏈合成的起始階段，）肽鏈的延伸，）肽鏈合成的終止與釋放。蛋白質(zhì)合成體系可以說是高度組織化的超分子體系，保證了蛋白質(zhì)生物合成的正確性和高效性。Translation is generally divided into three stages: initiation, elongation, and termination. In both prokaryotes and eukaryotes the

35、 first step of the initiation stage is the binding of a specific initiator methionyl tRNA and the mRNA to the small ribosomal subunit. The large ribosomal subunit then joins the complex, forming a functional ribosome on which elongation of the polypeptide chain proceeds. A number of specific nonribo

36、somal proteins are also required for the various stages of the translation process Figure. Overview of translation 肽鏈合成的起始階段的超分子體系 The first translation step in bacteria is the binding of three initiation factors (IF-1, IF-2, and IF-3) to the 30S ribosomal subunit. The mRNA and initiator N-formylmet

37、hionyl tRNA then join the complex, with IF-2 (which is bound to GTP) specifically recognizing the initiator tRNA. IF-3 is then released, allowing a 50S ribosomal subunit to associate with the complex. This association triggers the hydrolysis of GTP bound to IF-2, which leads to the release of IF-1 a

38、nd IF-2 (bound to GDP). The result is the formation of a 70S initiation complex (with mRNA and initiator tRNA bound to the ribosome) that is ready to begin peptide bond formation during the elongation stage of translation. Figure. Initiation of translation in bacteria Three initiation factors (IF-1,

39、 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, fol

40、lowed by the release of IF-1 and IF-2 bound to GDP. Initiation in eukaryotes is more complicated and requires at least ten proteins (each consisting of multiple polypeptide chains), which are designated eIFs (eukaryotic initiation factors). The factors eIF-1, eIF-1A, and eIF-3 bind to the 40S riboso

41、mal subunit, and eIF-2 (in a complex with GTP) associates with the initiator methionyl tRNA. The mRNA is recognized and brought to the ribosome by the eIF-4 group of factors. The 5 cap of the mRNA is recognized by eIF-4E. Another factor, eIF-4G, binds to both eIF-4E and to a protein (poly-A binding

42、protein or PABP) associated with the poly-A tail at the 3 end of the mRNA. Eukaryotic initiation factors thus recognize both the 5 and 3 ends of mRNAs, accounting for the stimulatory effect of polyadenylation on translation. The initiation factors eIF-4E and eIF-4G, in association with eIF-4A and eI

43、F-4B, then bring the mRNA to the 40S ribosomal subunit, with eIF-4G interacting with eIF-3. The 40S ribosomal subunit, in association with the bound methionyl tRNA and eIFs, then scans the mRNA to identify the AUG initiation codon. When the AUG codon is reached, eIF-5 triggers the hydrolysis of GTP

44、bound to eIF-2. Initiation factors (including eIF-2 bound to GDP) are then released, and a 60S subunit binds to the 40S 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 rib

45、osomal 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 identif

46、y 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 the

47、n joins the 40S complex. 肽鏈合成的延伸階段的超分子復(fù)合體 肽鏈延伸由許多循環(huán)組成，每加入一個(gè)氨基酸就是一次循環(huán)，每次循環(huán)包括三步：1）AA-tRNA與核糖體的結(jié)合，2）轉(zhuǎn)肽，3）移位。在每一循環(huán)步驟中，核糖體與各種延伸因子形成超分子體系，提供肽鏈延伸的空間和能量。另外，最近還發(fā)現(xiàn)RRF（ribosome recycling factor），它與延伸因子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