分子生物学(英文版)

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 repetitions 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 DNA 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 molecules 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 separates 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 half 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 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 Genesl 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 association 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”.在每一种生物中其单倍体基因组的DNA总量是特异的，被称为C值 (C Value)。 C值和生物结构或组成的复杂性不一致的现象称为C值悖论(C-value 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 hybridization (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 hairpin8. 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 materialThe 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 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 information 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 loss 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 components.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 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” somewhatGyrase-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 initiated 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 CircleUnwinding 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 of 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 translation.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 single-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 deoxynucleoside 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 polymerase 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 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 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 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的复制有DNA聚合酶及多种蛋白质因子参与，DNA聚合酶也有多种类型。其中DNA Pol及DNA Pol在细胞核内DNA的复制中起主要作用。DNA Pol催化前导链及滞后链的合成，是主要负责DNA复制的酶。DNA Pol的功能主要是引物合成。DNA Pol是线粒体中的复制酶。 Chapter 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 sequences 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 prokaryotes: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: it 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) 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 single 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 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 be 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 rRNA 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 an intron ( 14 nucleotides) in the anticodon loop by splicing. Replacing two U residues at the 3end by CCA, which is found in all mature tRNAs. Modifying some residues to characteristic bases, e.g., inosine(次黄嘌呤）, dihydrouridine and pseudouridine.4. Transcription in eukaryotes5 notable differences: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 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), or blocking (as a repressor) the recruitment of RNA polymerase to specific genes. Overview of the mRNA maturation stepsThree types of RNA polymerases function in eukaryotesClass IClass IIClass IIILocation:Nucleolus(核仁)Nucleoplasm(核质)Nucleoplasm(核质)Product:rRNAmRNAtRNA5S RNACapping mRNA 5 cap is 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 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 eukaryotic 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 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 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 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 ribonucleoproteins (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，在核仁中存在的一类小的RNA，主要为指导rRNA或snRNA中特异位点2-O-核糖甲基化修饰，假尿嘧啶化修饰，或作为分子伴侣参与靶标RNA高级结构的形成，如在核糖体RNA的加工过程中起作用。在剪接过程中，剪接装置中的各种snRNA间以及snRNA与底物间的碱基配对相互作用是非常重要的，这些作用可引起结构的变化以利于剪接的进行，使参与反应的基团处于合适的位置，并可能产生具有催化作用的活性中心。 自我剪接和借助于剪接体的mRNA剪接之间的区别是mRNA剪接依赖于核内小核糖核蛋白snRNP，snRNP是由核内小RNA（snRNA）和相关蛋白组成的。 存在5种snRNP：U1 snRNP，U2 snRNP，U5 snRNP和U4/U6 snRNP，它们与内含子形成剪接体（spliceosome）。Group I intron splicing mechanism (no lariat formed): Group II 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 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 named 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 Southerns name. As the technique was eponymously named, Southern blot should be capitalized, whereas northern and western blots should not. 将待检测的DNA分子用/不用限制性内切酶消化后，通过琼脂糖凝胶电泳进行分离，继而将其变性并按其在凝胶中的位置转移到硝酸纤维素薄膜或尼龙膜上，固定后再与同位素或其它标记物标记的DNA或RNA探针进行反应。如果待检物中含有与探针互补的序列，则二者通过碱基互补的原理进行结合，游离探针洗涤后用自显影或其它合适的技术进行检测，从而显示出待检的片段及其相对大小。Southern blot 是分析DNA的杂交技术，Northern blot是分析RNA的，而Western blot是分析蛋白质的 Southern 和Northerin原理基本相同，都是利用DNA或RNA的复性过程，但过程上也有区别，主要是 Southern是先电泳后变性，而Northern是先变性后电泳； Southern是碱变性，而Northern采用甲醛、乙二醛、二甲基亚砜等变性，因为采用碱变性会导致RNA水解 RNA的特点 T U 核糖 单链 核酶 RNA的转录合成从化学角度来讲类似于DNA的复制，多核苷酸链的合成都是以53的方向，在3-OH末端与加入的核苷酸磷酸二酯键，但是由于复制和转录的目的不同，转录又具有其特点： 对于一个基因组来说，转录只发生在一部分基因，而且每个基因的转录都受到相对独立的控制 转录是不对称的 转录时不需要引物，而且RNA链的合成是连续的 模板识别中真核与原核的不同真核生物RNA聚合酶不能直接识别基因的启动子区，需要转录调控因子按特定顺序结合于启动子上，RNA聚合酶才能与之结合形成前起始复合物。转录作用是RNA聚合酶催化的DNA指导的RNA合成作用。反应是以DNA为模板，以四种三磷酸核苷（NTP）即ATP、GTP、CTP及UTP为原料，各种核苷酸之间的3、5磷酸二酯键相连进行的聚合反应。合成反应的方向为53。反应体系中还有Mg2+、Mn2+等参与，反应中不需要引物参与。碱基互补原则为A-U、G-C，在RNA中U替代T与A配对。 RNA聚合酶缺乏35外切酶活性，所以没有校正功能。 RNA聚合酶大肠杆菌RNA聚合酶由五个亚基组成，为二个，一个，一个和一个因子，2四个亚基组成核心酶，加上因子后成为全酶。因子与核心酶的结合不紧密，容易脱落。RNA聚合酶亚基和亚基是酶的活性中心。 亚基可能与核心酶的组装及启动子识别有关。因子负责模板链的选择和识别DNA模板上转录的起始部位。 l 因子可以提高RNA聚合酶对启动子区的亲和力，降低RNA聚合酶对非专一性位点的亲和力l 不同的因子识别不同的启动子，调控不同基因转录的起始l 因子的释放表明转录起始的终止，酶-DNA-RNA形成稳定复合物，转录进入延伸期原核生物RNA聚合酶的几个特点：聚合速度比DNA复制的聚合反应速率要慢；缺乏35外切酶活性，无校对功能，RNA合成的错误率比DNA复制高很多；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，这是由于它们可以和RNA聚合酶的亚基相结合，而影响到酶的作用。启动子(promoter, P)是位于结构基因5端上游区的能被RNA聚