分子生物學(xué)(英文版)(共26頁)

1、精選優(yōu)質(zhì)文檔-傾情為你奉上Chapter 3 Nucleic Acid1. Physical and chemical structure of DNAl Double-stranded helixl Major groove and minor groovel Base pairingl The two strands are antiparallell G+C content (percent G+C)l Satellite DNASatellite DNA consists of highly repetitive DNA and is so called because repetit

2、ions of a short DNA sequence tend to produce a different frequency of the nucleotides adenine, cytosine, guanine and thymine, and thus have a different density from bulk DNA - such that they form a second or 'satellite' band when genomic DNA is separated on a density gradient. 2. Alternate D

3、NA structureTwo bases have been extruded from base stacking at the junction. The white line goes from phosphate to phosphate along the chain. O is shown red, N blue, P yellow and C grey.3. Circular and superhelical DNADNA can also form a double-stranded, covalently-closed circle. These circular mole

4、cules are often coiled into a superhelix, the formation of which is catalyzed by enzymes called topoisomerases.4. Denaturation of DNADenaturation: A transition from the native to the denatured stateDNA denaturation: also called DNA melting, is the process by which double-stranded DNA unwinds and sep

5、arates into single-stranded strands through the breaking of hydrogen bonding between the bases. Hyperchromicity / Hyperchromic effect: the striking increase in absorbance of DNA (A260) caused by the denaturation of the double-stranded DNA moleculeMelting temperature (Tm) : the temperature at which h

6、alf of the DNA strands are in the double-helical state and half are denatured. The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule. Factors Affecting Tml G-C content of samplel reagents that increase the solubility of

7、the bases (anything that disrupts H-bonds or base stacking)l Salt concentrationl pH l Length5. RenaturationStrands can be induced to renature (anneal) under proper conditions. Factors to consider:l Temperaturel Salt concentrationl DNA concentrationl TimeRepetitive Sequences l Unique: Single Copy Gen

8、esl Slightly repetitive (2-10 copies)l Middle repetitive (10- hundreds) -Clustered -Dispersedl Highly repetitive (hundreds to millions) -Short sequences in satellite DNA -Sequences of normal length in certain genes that exist in very large numbersC-value ParadoxThere is apparently a lack of associat

9、ion between C-value (the amount of DNA present in the haploid genome of different organisms )and the degree of organismal complexity of various multi-cellular organisms. In 1971, Thomas named this phenomenon, “C-value Paradox”.在每一種生物中其單倍體基因組的DNA總量是特異的，被稱為C值 (C Value)。 C值和生物結(jié)構(gòu)或組成的復(fù)雜性不一致的現(xiàn)象稱為C值悖論(C-va

10、lue paradox)。 6. HybridizationHybridization: the technique wherein renatured DNA is formed from separate single-stranded samples .Heteroduplexing: renaturation combined with electron microscopy in a procedure allows the localization of common, distinct,and missing sequences in DNA.DNA-RNA hybridizat

11、ion (Northern hybridization): the use of filter hybridization to detect sequence complementarity between a single strand of DNA and an RNA molecule.7. The structure of RNATypes: mRNA, tRNA, rRNADistinctions:- ribose replaces deoxyribose;- U replaces T;- Single-stranded Conformation: stem-loop or hai

12、rpin8. Hydrolysis of nucleic acidThe phosphodiester bonds of both DNA and RNA can be broken by hydrolysis either chemically or enzymatically.Ribozymes: the RNA enzymes, are able to cleave and form specific phosphodiester bonds in a manner analogous to protein enzymes.Chapter 6 The genetic materialTh

13、e Path to the Watson and Crick Model1928, Griffith, transformation in pneumococci（肺炎球菌）1944, Avery, Griffiths transforming principle was DNA1950, Chargaff, a pattern in the amounts of the four bases 1952, Hershey and Chase, DNA is the genetic material1953, Franklin, the x-ray picture of DNAChargaffs

14、 ruleIn the DNA of all species examined, A=T, G=CThe total amount of purines (A+G)=pyrimidines (T+C) in DNAThe ration of (A+T)/(G+C) varies from species to speciesDNA properties and functions1.DNA has the ability to store genetic information, which can be expressed in the cell as need.2.This informa

15、tion can be transmitted to daughter cells with minimal error. (This process requires complex enzymes and repair mechanisms.)3.DNA possesses both physical and chemical stability so information is not lost over long periods of time (years).4.DNA has the potential for heritable change without major los

16、s of parental information.DNA-genetic material: Double-stranded DNA has evolved as the genetic material because it is especially well-suited for replication, repair, occasional change, and long-time stability.Gene: Genes contain all the information for the synthesis and functioning of cellular compo

17、nents.Transcription: the process of synthesizing RNA molecules from a DNA template.Triplets / codons: the RNA nucleotide sequence is read (on ribosomes) in sequential groups of three bases.Mutation: the process by which a base-sequence changes.The central dogma: DNA makes RNA, makes protein.chapter

18、7 DNA replicationSemiconservative replication of double-stranded DNAUntwisting of highly coiled DNA is required for DNA replicationTopoisomerase Type I : Work ahead of replicating DNA Mechanism Makes a cut in one strand, passes other strand through it. Seals gap. Result: the DNA is “relaxed” somewha

19、tGyrase-A Type II Topoisomerase Introduces negative supercoils breaks both strands Section located away from actual cut is then passed through cut site. Initiation of DNA replication Replicaion initiated at specific sites: Origin of Replication (ori) Two Types of initiation: De novo Synthesis initia

20、ted with RNA primers. Most common. Covalent extensionsynthesis of new strand as an extension of an old strand (“Rolling Circle”). Limited to certain viruses.De novo Initiation Binding to Ori C by DnaA protein Opens Strands Replication proceeds bidirectionallyCovalent extension initiation Rolling Cir

21、cleUnwinding of DNA for replicationHelicase:n Breaks hydrogen bonds and eliminates hydrophobic interactionsn Needs energy supplied by ATPn Encoded by the DnaB gene in E.coliSingle-strand DNA binding proteins (SSB):Bind to the exposed strands, coat them and block the re-annealing process.Elongation o

22、f newly synthesized strands1.The polymerization reaction and the polymerasesEnzyme: polymerase IIINeeded: substrates, template, primerDirection: 5 32. Correcting mismatched basesThe 5-3 exonuclease activity of pol I at a single-strand break (nick) can occur simultaneously with polymerization-nick tr

23、anslation.DNA polymerase III consists of multiple subunitsn Pol I and pol III are both involved in E.coli DNA replication. Pol III is the major polymerase.n Both poly I and poly III possess a proofreading or editing function (3-5 exonuclease activity ).n The 5-3 exonuclease activity of pol I at a si

24、ngle-strand break (nick) can occur simultaneously with polymerization-nick translation.n DNA polymerase III consists of multiple subunits.n All known polymerases can work only in the 5-P 3-OH direction.Pol I and pol III have some features in common:l 5-3 polymerization activityThe four deoxynucleosi

25、de 5-triphosphatesA primer with a free 3-OHA templatel 3-5 exonuclease activityAntiparallel DNA strands and discontinuous replicationn The two strands of DNA is antiparallel and the replication is discontinuous synthesis.n A primer is required for chain initiation and two different enzymes (RNA poly

26、merase and primase) are known to synthesize primer RNA molecules.n DNA ligase joins precursor fragments and pol I as well as RNase H participates in the removal of primer.RNA polymerase: initiation of leading-strand synthesisPrimase: synthesis of primers for lagging-strandPrimosome: helicase/primase

27、 complexPol I: removal of the primer and replacement of DNADNA ligase: joining the fragment (gap sealed)The complete DNA replication systemBidirectional replication speeds up DNA synthesisReplication of eukaryotic chromosomes1. Eukaryotes have more and large chromosomes.2. Eukaryotic replication may

28、 require as much as 6-8 hours for completion versus the 40 minutes needed by E.coli.3. There are multiple, rather than a single, replication origins along eukaryotic chromosomes. They are spaced about 20 kb apart.4. Eukaryotic DNA replication is at the rate of about 10-100 nucleotides per second as

29、opposed to the prokaryotic rate of about 1500 nucleotides per second.5. At least five types of DNA polymerases have been found in eukaryotic cells. 真生物DNA的復(fù)制有DNA聚合酶及多種蛋白質(zhì)因子參與，DNA聚合酶也有多種類型。其中DNA Pol及DNA Pol在細(xì)胞核內(nèi)DNA的復(fù)制中起主要作用。DNA Pol催化前導(dǎo)鏈及滯后鏈的合成，是主要負(fù)責(zé)DNA復(fù)制的酶。DNA Pol的功能主要是引物合成。DNA Pol是線粒體中的復(fù)制酶。 Chapter

30、8 Transcription1. Enzymatic synthesis of RNAE. Coli RNA polymeraseHoloenzyme:core enzyme: 2factor(1) Binding of RNA pol to a template at specific site(2) Initiation(3) Chain elongation(4) Chain termination and release2. Transcription signalsIn prokaryotes, the promoter consists of two short sequence

31、s at -10 and -35 positions upstream from the transcription start site. l the -10 element :Pribnow box, usually consists TATAAT, is absolutely essential to start transcription in prokaryotes.l the -35 element :usually consists of TTGACA. Its presence allows a very high transcription rate. In prokaryo

32、tes:In eukaryotes:Termination Termination of RNA synthesis occurs at specific base-sequences in the DNA molecule, called terminators. Intrinsic terminators: rho-independent terminators, the termination sequences allow RNA polymerase to terminate elongation spontaneously. rho-dependent terminators: i

33、t is dependent on a specific protein called a rho factor. Intrinsic Termination RNA pol passes over inverted repeats Hairpins begin to form in the transcript Poly-U:poly-A stretch melts RNA pol and transcript fall offRho: Mechanism Rho binds to transcript at r loading site (up stream of terminator)

34、Hairpin forms, pol stalls Rho helicase releases transcript and causes termination3. Classes of RNA moleculesMessenger RNA: short lifetimeRibosomal RNATransfer RNA cistron: a DNA segment corresponding to one polypeptide chain plus the start and stop signalsmonocistronic mRNA: an mRNA encoding a singl

35、e polypeptidepolycistronic mRNA: an mRNA encoding several different polypeptide chainsRNA processing is to generate a mature mRNA (for protein genes) or a functional tRNA or rRNA from the primary transcript. Processing of pre-mRNA involves the following steps: Capping: add 7-methylguanylate (m7G) to

36、 the 5' end. Polyadenylation: add a poly-A tail to the 3' end. Splicing: remove introns and join exons. In some cases, RNA editing is also involved.Processing of pre-rRNA and pre-tRNA: The newly transcribed pre-rRNA is a cluster of three rRNAs: 18S, 5.8S and 28S in mammals.  They must b

37、e separated to become functional.  Pre-rRNA is synthesized in the nucleolus（核仁）.  The snRNA, and their associated proteins in the nucleolus are involved in the cleavage of the pre-rRNA. 5S rRNA is synthesized in the nucleoplasm.  It does not require any processing.   After 5S rRN

38、A is synthesized, it will combine with 28S and 5.8S rRNAs, forming the large subunit of the ribosome.Pre-tRNA requires extensive processing to become a functional tRNA.  Four types of modifications are involved: Removing an extra segment ( 16 nucleotides) at the 5' end by RNase P. Removing

39、an intron ( 14 nucleotides) in the anticodon loop by splicing. Replacing two U residues at the 3'end by CCA, which is found in all mature tRNAs. Modifying some residues to characteristic bases, e.g., inosine(次黃嘌呤）, dihydrouridine and pseudouridine.4. Transcription in eukaryotes5 notable differen

40、ces:l 3 classes of RNA pol for different classes of RNAl mRNA molecules are long livedl Both the 5 and 3 end are modifiedl Introns are excised and fragments are rejoined in mRNA processingl All eukaryotic mRNA are monocistronicInitiation of transcription in eukaryotesTATA box: -29Upstream activation

41、 sitesenhancersl Transcription factorsA transcription factor is a protein that binds to specific sequences of DNA and generally increase the transcription from vicinal promoters. Transcription factors perform this function alone, or with other proteins in a complex, by promoting (as an activator), o

42、r blocking (as a repressor) the recruitment of RNA polymerase to specific genes. Overview of the mRNA maturation stepsThree types of RNA polymerases function in eukaryotes Class IClass IIClass IIILocation:Nucleolus(核仁)Nucleoplasm(核質(zhì))Nucleoplasm(核質(zhì))Product:rRNAmRNAtRNA5S RNACapping mRNA 5 cap is

43、 a reversed guanosine residue so there is a 5-5 linkage between the cap and the first sugar in the mRNA. Guanosine cap is methylated. First and second nucleosides in mRNA may be methylated The function of 5 capPolyadenylation at the 3' endThe major signal for the 3' cleavage is the sequence

44、AAUAAA.  Cleavage occurs at 10-25 nucleotides downstream from the specific sequence.   A second signal is located about 50 nucleotides downstream from the cleavage site.   This signal is a GU-rich or U-rich region. Polyadenylation Polyadenylation occurs on the 3 end of virtually all e

45、ukaryotic mRNAs. Occurs after capping Catalyzed by polyadenylate polymerase Polyadenylation associated with mRNA half-life Histones not polyadenylatedEukaryotic primary transcripts contain intervening sequences (introns)Introns: Untranslated intervening sequences in the primary transcripts of higher

46、 eukaryotesExons: Translated sequencesRNA splicing ( Process ) : the excision of the introns and the formation of the final mRNA molecules by joining of the exonshnRNA (heterogeneous nuclear RNA): the precursor and partially processed mRNA moleculesSplice Site Recognition: Introns contain invariant

47、5-GU and 3-AG sequences at their borders (GU-AG Rule) Recognized by small nuclear ribonucleoprotein particles (snRNPs) that catalyze the cutting and splicing reactions. Internal intron sequences are highly variable even between closely related homologous genes. Alternative splicing allows different

48、proteins from a single original transcriptThere are 3 classes of splicing mechanisms: snRNP requiring self-splicing: group I and II, two transesterification reactions that are self-catalyzing tRNASplicing Overviewl Occurs in the nucleusl Splicing occurs on spliceosomes consist of small nuclear ribon

49、ucleoproteins (snRNPs) on splice sitel snRNPs contain small nuclear RNA (snRNA) and many types of snRNA have different functions in the splicing processsnoRNA：核仁小分子RNA，small nucleolar RNA，在核仁中存在的一類小的RNA，主要為指導(dǎo)rRNA或snRNA中特異位點(diǎn)2-O-核糖甲基化修飾，假尿嘧啶化修飾，或作為分子伴侶參與靶標(biāo)RNA高級結(jié)構(gòu)的形成，如在核糖體RNA的加工過程中起作用。在剪接過程中，剪接裝置中的各種sn

50、RNA間以及snRNA與底物間的堿基配對相互作用是非常重要的，這些作用可引起結(jié)構(gòu)的變化以利于剪接的進(jìn)行，使參與反應(yīng)的基團(tuán)處于合適的位置，并可能產(chǎn)生具有催化作用的活性中心。 自我剪接和借助于剪接體的mRNA剪接之間的區(qū)別是mRNA剪接依賴于核內(nèi)小核糖核蛋白snRNP，snRNP是由核內(nèi)小RNA（snRNA）和相關(guān)蛋白組成的。 存在5種snRNP：U1 snRNP，U2 snRNP，U5 snRNP和U4/U6 snRNP，它們與內(nèi)含子形成剪接體（spliceosome）。Group I intron splicing mechanism (no lariat formed): Group II

51、splicing mechanism: tRNAs: splicing reaction requires ATP and an endonuclease.  The endonuclease cleaves the phosphodiester bonds at both ends of the intron and the two exons are joined by a mechanism similar to the DNA ligation reaction 5. Means of studying intracellular RNAA Southern blot is

52、a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization. The method is name

53、d after its inventor, the British biologist Edwin Southern. Other blotting methods (northern blot , western blot) that employ similar principles, but using RNA or protein, have later been named in reference to Southern's name. As the technique was eponymously named, Southern blot should be capit

54、alized, whereas northern and western blots should not. 將待檢測的DNA分子用/不用限制性內(nèi)切酶消化后，通過瓊脂糖凝膠電泳進(jìn)行分離，繼而將其變性并按其在凝膠中的位置轉(zhuǎn)移到硝酸纖維素薄膜或尼龍膜上，固定后再與同位素或其它標(biāo)記物標(biāo)記的DNA或RNA探針進(jìn)行反應(yīng)。如果待檢物中含有與探針互補(bǔ)的序列，則二者通過堿基互補(bǔ)的原理進(jìn)行結(jié)合，游離探針洗滌后用自顯影或其它合適的技術(shù)進(jìn)行檢測，從而顯示出待檢的片段及其相對大小。Southern blot 是分析DNA的雜交技術(shù)，Northern blot是分析RNA的，而Western blot是分析蛋白質(zhì)的 S

55、outhern 和Northerin原理基本相同，都是利用DNA或RNA的復(fù)性過程，但過程上也有區(qū)別，主要是 Southern是先電泳后變性，而Northern是先變性后電泳； Southern是堿變性，而Northern采用甲醛、乙二醛、二甲基亞砜等變性，因?yàn)椴捎脡A變性會(huì)導(dǎo)致RNA水解 RNA的特點(diǎn)§ T U§ 核糖§ 單鏈§ 核酶 RNA的轉(zhuǎn)錄合成從化學(xué)角度來講類似于DNA的復(fù)制，多核苷酸鏈的合成都是以53的方向，在3-OH末端與加入的核苷酸磷酸二酯鍵，但是由于復(fù)制和轉(zhuǎn)錄的目的不同，轉(zhuǎn)錄又具有其特點(diǎn)： 對于一個(gè)基因組來說，轉(zhuǎn)錄只發(fā)生在一部分基因，而且

56、每個(gè)基因的轉(zhuǎn)錄都受到相對獨(dú)立的控制 轉(zhuǎn)錄是不對稱的 轉(zhuǎn)錄時(shí)不需要引物，而且RNA鏈的合成是連續(xù)的 模板識(shí)別中真核與原核的不同真核生物RNA聚合酶不能直接識(shí)別基因的啟動(dòng)子區(qū)，需要轉(zhuǎn)錄調(diào)控因子按特定順序結(jié)合于啟動(dòng)子上，RNA聚合酶才能與之結(jié)合形成前起始復(fù)合物。轉(zhuǎn)錄作用是RNA聚合酶催化的DNA指導(dǎo)的RNA合成作用。反應(yīng)是以DNA為模板，以四種三磷酸核苷（NTP）即ATP、GTP、CTP及UTP為原料，各種核苷酸之間的3、5磷酸二酯鍵相連進(jìn)行的聚合反應(yīng)。合成反應(yīng)的方向?yàn)?3。反應(yīng)體系中還有Mg2+、Mn2+等參與，反應(yīng)中不需要引物參與。堿基互補(bǔ)原則為A-U、G-C，在RNA中U替代T與A配對。 RN

57、A聚合酶缺乏35外切酶活性，所以沒有校正功能。 RNA聚合酶大腸桿菌RNA聚合酶由五個(gè)亞基組成，為二個(gè)，一個(gè)，一個(gè)和一個(gè)因子，2四個(gè)亞基組成核心酶，加上因子后成為全酶。因子與核心酶的結(jié)合不緊密，容易脫落。RNA聚合酶亞基和亞基是酶的活性中心。 亞基可能與核心酶的組裝及啟動(dòng)子識(shí)別有關(guān)。因子負(fù)責(zé)模板鏈的選擇和識(shí)別DNA模板上轉(zhuǎn)錄的起始部位。 l 因子可以提高RNA聚合酶對啟動(dòng)子區(qū)的親和力，降低RNA聚合酶對非專一性位點(diǎn)的親和力l 不同的因子識(shí)別不同的啟動(dòng)子，調(diào)控不同基因轉(zhuǎn)錄的起始l 因子的釋放表明轉(zhuǎn)錄起始的終止，酶-DNA-RNA形成穩(wěn)定復(fù)合物，轉(zhuǎn)錄進(jìn)入延伸期原核生物RNA聚合酶的幾個(gè)特點(diǎn)：聚合速度比DNA復(fù)制的聚合反應(yīng)速率要慢；缺乏35外切酶活性，無校對功能，RNA合成的錯(cuò)誤率比DNA復(fù)制高很多；原核生物RNA聚合酶的活性可以被利福霉素及利福平所抑制，這是由于它們可以和RNA聚合酶的亞基相結(jié)合，而影響到酶的作用。啟動(dòng)子(promoter, P)是位