分子生物学杨洋课程总结

1、the revised central dogmatranslationrna processingdna repair基基因因组组的的保保持持基基因因组组的的表表达达regulation分子生物学技术分子生物学技术（原理及应用）（原理及应用）课程总结课程总结 第一章第一章-绪论，绪论，dna和和rna结构结构 第二章第二章-染色体，染色质和核小体染色体，染色质和核小体(chromosomes, chromatin, and the nucleosome） 第三章第三章-dna的复制的复制(the replication of dna） 第四章第四章-dna的突变和修复的突变和修复(the m

2、utability and repair of dna） 第五章第五章-转录机制转录机制(mechanisms of transcription) 第六章第六章 rna剪接剪接(rna splicing） 第七章第七章 翻译翻译-1，2, 3 第八章第八章-遗传密码遗传密码 (genetic code) 第九章第九章-分子生物学技术分子生物学技术(techniques of molecular biology) 第十章第十章 原核生物基因表达调控原核生物基因表达调控(regulation in prokaryotes) 第十一章第十一章 真核生物基因表达调控真核生物基因表达调控(regulat

3、ion in eukaryotes) 第十二章第十二章-dna重组重组第一章第一章-绪论，绪论，dna和和rna结构结构 分子生物学的含义分子生物学的含义 分子生物学的发展分子生物学的发展 分子生物学研究内容分子生物学研究内容 what is molecular biology?define in broadly: （广义定义）（广义定义）understand biological phenomena in molecular terms (difficult to distinguish from biochemistry)define in restrictively: （狭义定义）（狭义

4、定义）the study of gene structure and their activities in molecular level（分子水平上研究基因的结构和功能）（分子水平上研究基因的结构和功能）gene concept gene structuregene replication gene expression gene recombinationgene mutationmolecular biology of gene（基因的分子生物学）（基因的分子生物学）第二章第二章-染色体，染色质和核小体染色体，染色质和核小体(chromosomes, chromatin, and th

5、e nucleosome） chromosome sequence & diversity（染色体序列和多样性）（染色体序列和多样性） the nucleosome（核小体）（核小体） higher-order chromatin structure（染色质的高级结构）（染色质的高级结构） regulation of chromatin structure（染色质结构的调控）（染色质结构的调控）nucleosomes are the building blocks of chromosomes（核小体是染（核小体是染色体的结构单位）色体的结构单位） 在真核细胞中大多数在真核细胞中大多数dna被

6、包装进被包装进核小体核小体 the nucleosome is composed of a core of eight histone proteins and the dna (core dna, 147 bp，核心，核心dna) wrapped around them（核小体由（核小体由8个组蛋白所形成的核组成，个组蛋白所形成的核组成，dna缠绕在组蛋白核上）缠绕在组蛋白核上） the dna between each nucleosome is called a linker dna（连接（连接dna). each eukaryote has a characteristic avera

7、ge linker dna length (20-60 bp)figure 7-18 dna packaged into nucleosomesix-fold dna compactiondna盘绕在组蛋白核上盘绕在组蛋白核上（h2a, h2b, h3，h4）第三章第三章-dna的复制的复制(the replication of dna） 1. the chemistry of dna synthesis(dna合成合成的化学基础）的化学基础） 2. the mechanism of dna polymerase（dna聚合酶的作用机制）聚合酶的作用机制） 3. the specializat

8、ion of dna polymerases（dna聚合酶的特化）聚合酶的特化） 4. the replication fork（复制叉）（复制叉） 5. dna synthesis at the replication fork（复（复制叉上的制叉上的dna合成）合成） 6. initiation of dna replication（复制的起始）（复制的起始） 7. binding and unwinding（结合和解旋）（结合和解旋） 8. finishing replication（复制的终止）（复制的终止）the chemistry of dna synthesis: substra

9、te, direction and energy. the mechanism of dna polymerase: 1 polymerization mechanism, 2 different ways of discriminating substrates, 2 catalytic sites; 3 domains.the specialization of dna polymerasesthe replication fork: the enzyme/proteins required to synthesize the leading and lagging strands.dna

10、 synthesis at the replication fork: holoenzyme/trombone model to explain how the anti-parallel template strands are copied/replicated toward the replication fork. replisome/protein interaction.summaryinitiation of dna replication/binding and unwinding: the replicon model; initiation in bacteria; ini

11、tiation control in eukaryotes-a link with cell cycle (pre-rc assembly and activation). finishing replication: telomeres, telomerase dna polymerase the polymerase chain reaction(pcr) is to used to amplify a sequence of dna in vitro, using a pair of primers each complementary to one end of the the dna

12、 target sequence.复制理论复制理论实验技术实验技术denaturation (变性变性): the target dna (template) is separated into two stands by heating to 95primer annealing (退火退火): the temperature is reduced to around 55 to allow the primers to anneal.polymerization (elongation, extension) (延伸延伸): the temperature is increased to

13、72 for optimal polymerization step which uses dntps and requires mg+.the principle of pcr:three different steps proceed in each pcr cycle. reverse transcriptase (rt)-pcr逆转录逆转录pcraaa(a)n5-capmrna(dt)1218 primeranneal5-capaaa(a)n35reverse transcriptiondntp, rt5-capaaa(a)n5cdna:mrna hybridregularpcr第四章

14、第四章-dna的突变和修复的突变和修复(the mutability and repair of dna）replication errors and their repair (复复制错误及其修复）制错误及其修复）dna damage （dna损伤）损伤）repair of dna damage（dna损伤的修损伤的修复）复） 复制中产生的错误以及它们是如何被修复的复制中产生的错误以及它们是如何被修复的 自发产生或来自外界的攻击造成的各种损伤自发产生或来自外界的攻击造成的各种损伤 细胞修复损伤的多种修复机制细胞修复损伤的多种修复机制生物在保持遗传物质忠实性方面的努力生物在保持遗传物质忠实性方

15、面的努力复制中产生的错误以及它们是如何被修复的复制中产生的错误以及它们是如何被修复的自发产生或来自外界的攻击造成的各种损伤自发产生或来自外界的攻击造成的各种损伤dna损伤的各种类型损伤的各种类型细胞修复细胞修复dna损伤的多种修复机制：损伤的多种修复机制： 1. direct reversal of dna damage by photoreactivation (光活化作光活化作用用) and alkyltransferase (烷基转移酶烷基转移酶)-直接将损伤逆转直接将损伤逆转 2. 剪切修复系统（剪切修复系统（excision repair system)： 仅仅将受损的核苷酸去除仅仅

16、将受损的核苷酸去除 将包含损伤的一小段单链将包含损伤的一小段单链dna去除去除 3. 重组修复系统（重组修复系统（recombination repair）：当）：当dna两条链都受损两条链都受损（断裂）时采用这种修复（断裂）时采用这种修复双链断裂修复（双链断裂修复（ double-strand break repair） 4. 移损移损dna合成（合成（translesion dna synthesis）：）：dna聚合酶的复聚合酶的复制进程被受损碱基阻碍时采用的修复制进程被受损碱基阻碍时采用的修复1. direct reversal of dna damage by photoreacti

17、vation (光活化作用光活化作用) and alkyltransferase (烷基转移酶烷基转移酶)-直接将损伤逆转直接将损伤逆转2. 剪切修复系统（剪切修复系统（excision repair system)：另一条未：另一条未受损的链作为模板，以便受损的链作为模板，以便dna聚合酶重新掺入正确的核苷酸聚合酶重新掺入正确的核苷酸仅仅将受损的核苷酸去除仅仅将受损的核苷酸去除将包含损伤的一小段单链将包含损伤的一小段单链dna去除去除3. 重组修复系统（重组修复系统（recombination repair）：当）：当dna两条链都受损（断裂）时采用这种修复两条链都受损（断裂）时采用这种修复

18、双链断裂修复双链断裂修复（ double-strand break repair）4. 移损移损dna合成（合成（translesion dna synthesis）：）：dna聚合酶的复制进程被受损碱基阻碍时采用的修复聚合酶的复制进程被受损碱基阻碍时采用的修复mechanisms to repair a damage（dna损伤的修复系统）损伤的修复系统）第五章第五章-转录机制转录机制(mechanisms of transcription)1. rna polymerase and the transcription cycle（rna聚合酶和转录周期）2. the transcripti

19、on cycle in bacteria（细菌的转录周期）3. transcription in eukaryotes（真核生物的转录）1. rna polymerases (rnap, 真核和原核的异同真核和原核的异同) and transcription cycle (initiation, elongation and termination)2. transcription cycle in bacteria: -initiation: (1) the feature of s s70 promoters. (2) promoter binding by s s70 transcrip

20、tion factor (recognition mechanism). (3) transition to open complex. (4) promoter escape and transition to the ternary complex. -elongation and editing by polymerase-termination: rho-independent and rho-dependent mechanism.3. transcription in eukaryotes by rnap ii: -initiation(i) cis-acting elements

21、: core promoter & regulatory sequences(ii) formation of the pre-initiation complex(iii) promoter escape and the ctd tail(iv) the function of each gtf (in vitro)(v) additional proteins for in vivo transcription. -elongation and proofreading involve a new set of gtfs -coupled with rna processing -5 ca

22、pping and 3 polyadenylation (mechanisms)-termination4. transcription in eukaryotes by rnap i and iii-initiation: promoter binding and formation of the closed complex.第六章第六章 rna剪接剪接(rna splicing）figure 13-1primary transcript外显子外显子内含子内含子前前mrna剪接后的剪接后的mrna rna剪接的化学基础剪接的化学基础 剪接体剪接体 剪接过程剪接过程 可变剪接可变剪接 自剪接

23、内含子自剪接内含子 rna编辑编辑 mrna转运转运1. why rna splicing is important? 2. chemical reaction: determination of the splice sites, the products, trans-splicing3. spliceosome: splicing pathway and finding the splice sites.4. self-splicing introns and mechanisms5. alternative splicing and regulation6. two different

24、 mechanisms of rna editing7. mrna transport-a link to translation 第七章第七章 翻译翻译-1，2, 3topic 1-4: four components of translation machinerymrna trna attachment of amino acids to trna (aminoacyl-trna synthetases) ribosometopic 5-7: translation processinitiation; elongation; terminationtopic 8: antibiotic

25、s ( (抗生素）抗生素）and translation2021-10-2129basic machinery of translation1.mrnas (5% of total cellular rna)-message rna2.trnas (15%)-transfer rna3.aminoacyl-trna synthetases (氨酰trna合成酶)4.ribosomes (核糖体): rrnaoverview of the events of translationtermination elongation initiationthree events must occur:1

26、. ribosome must be recruited to mrna2. a charged trna must be placed into the p site of ribosome3. the ribosome must be precisely positioned over the start codonthree initiation factors (if) direct the assembly of an initiation complex that contains mrna and initiator trna translation initiation fac

27、tors (if):if1: prevent trna from binding to the a site in the small subunitif2: a gtpase that interacts with small subunit, if1 and initiator trna (fmet-trnafmet); if2 can facilitate the association of fmet-trnafmet with the small subunitif3: binds to the small subunit and blocks it from reassociati

28、ng with large subunit, helps to dissociate the 70s ribosome into its large and small subunit进位进位易位易位肽键形成肽键形成two classes of release factors class i: recognize the stop codon and trigger hydrolysis of the polypeptide from peptidyl-trna prokaryotes: rf1-uag,uaa rf2-uga,uaa eukaryotes: erf1-uag,uga,uaa

29、class ii: stimulate the dissociation of the class i factors from the ribosome after release of polypeptide prokaryotes: rf3 eukaryotes: erf3 gtp-binding protein antibiotics ( (抗生素）抗生素）and translationpuromycin (嘌呤霉素嘌呤霉素)puromycin resembles the aminoacylated-trna, so it can bind to the a site of ribos

30、ome puromycin (嘌呤霉素嘌呤霉素)肽酰嘌呤霉素肽酰嘌呤霉素(peptidyl-puromycin)puromycin第八章第八章-遗传密码遗传密码 (genetic code)1 遗传密码的破译遗传密码的破译2 遗传密码的特征与性质遗传密码的特征与性质3 遗传密码的简并性和摆动假说遗传密码的简并性和摆动假说4 改变遗传密码的几类突变改变遗传密码的几类突变 4.1 错义突变错义突变 4.2 无义突变无义突变 4.3 移码突变移码突变5 基因内抑制突变和基因间抑制突变基因内抑制突变和基因间抑制突变6 遗传密码的通用性遗传密码的通用性“the genetic code is degen

31、erate” what does it mean? what are the benefits?what is the wobble concept? how the wobble in the anticodon affect the number of trnas to recognize the 61 codons?what are the mutations altering genetic code? what are suppressor mutations? what is the difference between intragenic suppression and int

32、ergenic suppression?what are the benefits of the code universality? intergenic suppression involves mutant trnasmutant trna genes suppress the effects of nonsense mutations in protein-coding genes. they act by reading a stop codon as if it were a signal for a specific amino acid.第九章第九章-分子生物学技术分子生物学技

33、术(techniques of molecular biology)dna-basic principlebasic procedure application1.separation by electrophoresis (电泳分离电泳分离) 2.cut by restriction endonuclease (限制性限制性内切酶切割技术内切酶切割技术)3.dna cloning and gene expression (基因基因克隆和表达技术）克隆和表达技术）4.分子杂交技术（分子杂交技术（ southern northern 5.pcr技术（聚合酶链式反应）技术（聚合酶链式反应）1. r

34、estriction digestion of your insert and vector using the same enzyme.2. use ligase (连接酶）连接酶） to join your insert and vector together.3. transform the ligation products into e. coli competent cells.4. select the desired clones: grow the cells on a plate containing tetracycline (四环素四环素).ecori1.protein

35、 purification (蛋白质纯化蛋白质纯化) 2.affinity chromatography can facilitate more rapid protein purification (亲和层析亲和层析纯化纯化)3.protein separation by page gel electrophoresis (蛋白质分离蛋白质分离) and identification by western analysis(免疫免疫印迹印迹) （一）dna-protein affinity chromatography (dna-蛋白亲和层析) (二）gel retardation (凝胶阻

36、滞, emsa，竞争性 emsa) （三）dnase i footprinting (dnase i 足迹法) （四）染色质免疫共沉淀技术（chromatin immunoprecipitation assay, chip） -研究体内dna与蛋白质相互作用的方法 （五）yeast one-hybrid system(酵母单杂交)- yeast two-hybrid system(酵母双杂交酵母双杂交) 免疫共沉淀技术免疫共沉淀技术targetbait no transcriptiontargetbait xybaitpredatorcdna for xyeast plasmidexpress

37、ion vectortransformedyeastfishing with the yeast two-hybrid systembait targetdoes x bindwith a unknownprotein?protein xbaittranscription machineryxybaitpreylac 2-galactosidasex-galblue colorbdad dna-bindingdomainbdxbdcdna library+ad fusion plasmid transformlibraries of dna molecules can be created b

38、y cloning (genomic library and cdna library) (dna文库文库) is a population of identical vectors that each contains a different dna insert. (基因组文库基因组文库) : the dna inserts in a dna library is derived from restriction digestion of the genomic dna. (cdna文库文库) : the dna inserts in a dna library is converted

39、from the mrnas of a tissue, a cell type or an organism. cdna stands for the dna copied from mrna. proteomics (蛋白质组学蛋白质组学)three principle methods 1. 2-d gel electrophoresis for (蛋白质分离蛋白质分离). 2. ms spectrometry for the precise of the molecular weight and identify of a protein (蛋白质鉴定蛋白质鉴定). 3. bioinfor

40、matics for proteins and peptides to the predicted protein coding sequence in the genome (蛋白质蛋白质确定确定). 原理机制原理机制 应用应用rna干扰技术干扰技术（rna interference, rnai） 第十章第十章 原核生物基因表达调控原核生物基因表达调控(regulation in prokaryotes) topic 1 principles of transcriptional regulation topic 2 regulation of transcription initiatio

41、n: examples from bacteria (lac operon,乳糖操纵子乳糖操纵子) topic 3 examples of gene regulation after transcription initiation (trp operon，色氨酸操纵子，色氨酸操纵子) （基于可变（基于可变s s因子的全局性调控）因子的全局性调控）topic 1 principles of transcriptional regulation gene expression is controlled at different stages (基基因表达调控可以发生在不同时期因表达调控可以发生

42、在不同时期): the key step of gene regulation takes place at the initiation of transcription gene expression is controlled by regulatory proteins (调控蛋白调控蛋白) positive regulators or activators negative regulators or repressors recruitment regulation (招募调控招募调控) allostery regulation (异构调控异构调控): converts the c

43、losed complex to open complex basic principle of regulation of gene expression: interaction between trans-acting factor (regulatory protein) and cis-acting element (control element):反式反式作用因子和顺式作用元件作用因子和顺式作用元件 control elementstructural genesbasic principle of regulation of gene expression:interaction

44、 between trans-acting factor (regulatory protein) and cis-acting element (control element) 反式作用因子和顺式作用元件之间的相互作用反式作用因子和顺式作用元件之间的相互作用trans-acting factortopic 2 regulation of transcription initiation: examples from bacteria (lac operon) structural genes control elements, such as operator(sequence regul

45、ator genea unit of prokarytoic gene expression and regulation which typically includes: 1. structural genes for enzymes in a specific biosynthetic pathway whose expression is coordinately controlled 2. control elements, such as operator(sequence-cis-acting element 3. regulator gene(s) code for regul

46、atory protein which can recognize and interact with the control elements negative control of the lac operon: interaction between repressor and operator (乳糖操纵子负调控乳糖操纵子负调控)two key elements: laci: encoding lac repressor, trans-acting factor operator: lac repressor binding site, cis-acting elementlacipr

47、omoterrna polymeraselaczlacylacano transcriptiontranscriptionoperatoractive repressorinactive repressor阻遏阻遏去阻遏去阻遏 positive control of the lac operon: interaction between cap-camp complex and cap binding site (乳糖操纵子正调控乳糖操纵子正调控) cap (catabolite activator protein, 代谢产物代谢产物激活蛋白激活蛋白) or crp (camp recepto

48、r protein, camp受体蛋白受体蛋白) the cap-camp binding sitemechanismcap-campcap binding sitelacztranscription（转录）（转录）促进了促进了rna聚合酶和启动子结合形成稳定的开放复合体聚合酶和启动子结合形成稳定的开放复合体(rna聚合酶聚合酶)(启动子启动子)the lac operon topic 3 examples of gene regulation after transcription initiation (trp operon)two layers of regulation are inv

49、olved: (1) transcription repression by the trp repressor (阻遏作用阻遏作用)-粗调粗调(2) attenuation(衰减作用衰减作用)-细调细调transcription repression by the trp repressor (阻遏作用阻遏作用) -粗调粗调低色氨酸低色氨酸: 去阻遏去阻遏高色氨酸高色氨酸: 阻遏阻遏inactive repressoractive repressorno transcription色氨酸色氨酸14个氨基酸的前导肽个氨基酸的前导肽low trphigh trpfig 16-21compleme

50、ntary 2:3 elongation of transcription complementary 3:4 termination of transcription （基于可变（基于可变s s因子的全局性调控）因子的全局性调控） 细菌经历逆境：饥饿，热激，缺氮；枯草芽孢细菌经历逆境：饥饿，热激，缺氮；枯草芽孢杆菌的孢子形成等杆菌的孢子形成等细菌通过转录的全局性变化对其环境变化作出细菌通过转录的全局性变化对其环境变化作出应答，这些全局性的转录变化是通过应答，这些全局性的转录变化是通过rna聚合聚合酶的变化得以实现的酶的变化得以实现的 rna聚合酶的变化本质上是因子的变化因子的变化， 因子控制

51、转录的特异性细菌通过转录的全局性变化对其环境变化作出细菌通过转录的全局性变化对其环境变化作出应答，这些全局性的转录变化是通过应答，这些全局性的转录变化是通过rna聚合聚合酶的变化得以实现的酶的变化得以实现的第十一章第十一章 真核生物基因表达调控真核生物基因表达调控(regulation in eukaryotes) topic 1: principles of regulation in eukaryotes topic 2transcription factors topic 3: chromatin structure and its effect on transcription top

52、ic 4 repressorstopic 1 principles of regulation in eukaryotes similarity of regulation between eukaryotes and prokaryote difference in regulation between eukaryotes and prokaryotedifference in regulation between eukaryotes and prokaryote1. pre-mrna splicing adds an important step for regulation. (in

53、tron, extron, mrna前体的剪接前体的剪接)2. the eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart. (真核转录机器更复杂真核转录机器更复杂, transcription factors)3. nucleosomes and their modifiers influence access to genes. (核小体及其修饰体核小体及其修饰体) 4. many eukaryotic genes have more regulatory binding

54、 sites and are controlled by more regulatory proteins than are bacterial genes. (真核基因有更多真核基因有更多调控蛋白调控蛋白结合位点结合位点) topic 2 transcription factorsgeneral transcription factors: basal level transcriptiongene-specific transcription factors (activator) eukaryotic activators (真核激活蛋白真核激活蛋白) have separate dna

55、 binding and activating functions: contains separate dna binding and activating domains the based on the separation of dna binding and activating domains is used to identify proteins interacting with each othereukaryotic activators (真核激活蛋白真核激活蛋白) also work by recruiting (招募招募) as in bacteria eukaryo

56、tic activators recruit polymerase indirectly in two ways:1. interacting with parts of the transcription machinery except for rna polymerase.2. recruiting nucleosome modifiers that alter chromatin in the vicinity of a gene.the contains polymerase and numerous proteins being organized to several compl

57、exes, such as the mediator and the tfd complex. activators interact with one or more of these complexes and recruit them to the promoter.figure 17-9 nucleosomes are the building blocks of chromosomes histones are small, positively charged (basic) proteins the core histones each have an n-terminal “t

58、ail”, the sites of extensive modifications histone not only is the structural component of nucleosome, but also can regulate the gene expression histone modification such as acetylation（组蛋白乙酰化酶）（组蛋白乙酰化酶） repressorsin eukaryotes, most repressors repress transcription by binding to sites that overlap

59、with the promoter and thus block binding of polymerase. (bacteria often do so)commonly, eukaryotic repressors that compact the nucleosome or remove the groups recognized by the transcriptional machinery contrast to the activator recruited nucleosome modifers, histone deacetylases (组蛋白去乙酰组蛋白去乙酰化酶化酶)

60、repress the transcription by removing the acetyl groupssome modifier adds methyl groups to the histone tails, which frequently repress the transcription.this modification causes transcriptional silencing.three other ways in which an eukaryotic repressor works include: . (2)binds to a site different