分子生物学英文课件：chapter1 Structure and Function of Nucleic Acid (2)

1、,Molecular Biology,Contents of molecular biology,Nucleic acid and nucleotides Gene expression Synthesis of DNA (replication) Synthesis of RNA (transcription) Synthesis of protein (translation) Regulation of gene expression Genetic engineering Signal transduction,Research History of Molecular Biology

2、,In 1869, F.Miescher: nuclein In 1928, Fred Griffith: transformation In 1944, Avery and his colleges: DNA genetic material In 1953, Watson and Crick: DNA double helical model In 1957, Kornberg: DNA polymerase In 1958, Crick: the central dogma In 1961, Jacob and Monod : operon theory In 1966,Nirenber

3、g、Ochoa and Khorana: the genetic codon In 1970, Temin and Baltimore: reverse transcriptase,some substance from the heat-killed S cells had been picked up by some of the R cells, changing them into virulent S type cells. Griffith coined the term transformation to describe this process.,1928 by Fred G

4、riffith,In 1967-1970, R.Yuan and H.O.Smith: Restriction endonuclease In 1975-1977, Sanger、Maxam and Gilbert: DNA sequencing In 1990s, automatic DNA sequencing In 1981, Cech: rRNA self splicingribozyme In 1981, the first transgenic mice In 1985, Mullis: PCR In 1990, HGP launched In 1990, ADA deficien

5、cy gene therapy In 1996, “Dolly” sheep In 1998, AndrewZ.Fire and Craig C.Mello : RNAi (2006 Nobel prize) In 2001, Publishing of the draft version of the Human Genome,Transgenic Mouse,1982,Achievements of gene engineering,Transgenic plant,克隆羊“多莉”,克隆羊“多莉”的研究者 伊恩维尔穆特（Wilmut）,Nucleus transferring,clonin

6、g,Biochip,Human Genome Project,DNA,RNA,Protein,The Central Dogma,Replication,Transcription,Translation,Reverse transcription,DNA and protein,Chapter 1 structure and function of nucleic acid,Nucleic acids are biological information macromolecules,Classification: Deoxyribonucleic acid, DNA: present in

7、 nucleus and mitochondria; store and carry genetic information. Ribonucleic acid, RNA: present in cytoplasm and nucleus; involved in expression of genetic information.,Section I Chemical composition and primary structure of nucleic acid,Nucleotides are basic component units of nucleic acid,(RNA),(DN

8、A),nucleotide,Base Pentose phosphate,Base,(Only in DNA) (Only in RNA),I. Chemical composition of nucleotides,酮式烯醇式互变异构,胺式亚胺式互变异构,Pentose: ribose in RNA, deoxyribose in DNA,The principal derivatives of purines and pyrimidines are nucleosides and nucleotides,Nucleoside= base (Purine or a Pyrimidine) +

9、pentose (ribose or deoxyribose) ribose ribonucleoside 2-deoxyribose deoxyribonucleoside purine purine nucleoside Pyrimidine pyrimidine nucleoside,Base and ribose(deoxyribose) are linked via a glycosidic bond :,AR CR,Nucleotide = nucleoside + phosphate or = Base ( purine or pyrimidine) + pentose ( ri

10、bose or deoxyribose) + phosphate ribose ribonucleotide 2-deoxyribose deoxyribonucleotide Purine purine nucleotide Pyrimidine pyrimidine nucleotide,Nucleotides are phosphorylated nucleosides,Ester bond,Nucleosides and phosphates are linked by ester bond:,Ester bond,glycosidic bond,Types of ribonucleo

11、tides and deoxyribonucleotides,Derivatives of nucleotide,II. primary structure of nucleic acid The primary structure is the sequence of nucleoside monophosphates from 5 end to 3 end in nucleic acid . (usually written as the sequence of bases).,Phosphodiester bond,Written from 5 to 3:,The differences

12、 between DNA and RNA:,Section II Spatial structure and function of DNA,Chargaffs Rules: purines=pyrimidines A=T G=C X-ray diffraction studies of Franklin and Wilkins Helix molecule Double strands,Backgrounds of proposition of double helix,I. The secondary structure of DNA double helix model,5,5,3,3,

13、Minor groove,Major groove,Antiparallel complementory double helix strand. Base pairing: A-T C-G Pentose-phosphate backbone outside and base pairs inside. Stabilized by hydrogen bond and base stacking force. genetic information consists in the DNA base sequences,1. Two polynucleotide chains , right-h

14、anded double helix. 2.The two strands of DNA are antiparallel. 3.Pentose-phosphate backbone outside and base pairs inside 4.The surface of the double helix contains two grooves : the major and minor grooves. 5. Each base is hydrogen bonded to a base in the opposite strand to form a base pair. The pl

15、anes of base pair are perpendicular to its axis. (A pairs with T; G pairs with C) 6. the diameter is 2.37 nm, the pitch of a turn is 3.54 nm, 10.5 base pairs per turn, the distance between two base planes is 0.34 nm. 7. stabilizing force : hydrogen bond and base stacking force,Points of DNA double h

16、elical model:,Different types of DNA:,The differences of three types of DNA,Triplex DNA,In these triple helices, the third strand remains associated with duplex DNA through non-Watson-Crick interactions now known as Hoogsteen pairing.,N,6,Structure of a G-quadruplex. Left: a G-tetrad. Right: an intr

17、amolecular G-quadruplex,II. Supercoiled structure of DNA in prokaryotes,relaxed,supercoiled,Telephone cord,III. nucleosome in eukaryotes,nucleosome,Nucleosome: The binding of DNA by the histones creates a structure unit of chromatin, the structure unit is called nucleosome.,Nucleosome,linker,Nucleos

18、ome core,Histone H1 60bp of DNA,Octamer of histone (two molecules each of H2A 、H2B、H3、H4) 150bp of DNA,H1 histone,IV. Function of DNA:,*DNA is carrier of genetic information. It can transfer genetic information by replication. * Gene：In molecular terms, it is a functional segment of DNA that codes f

19、or polypeptide chain or RNA molecules. * Genome is the total genetic information of an organism. For most organisms, it is the complete DNA sequence.,The chemical nature of RNA differs from that of DNA,In RNA, the sugar is ribose rather than 2-deoxyribose of DNA. Instead of thymine, RNA contains the

20、 uracil. RNA exists as a single strand, whereas DNA exists as a double-stranded helical molecule. In RNA, guanine content cytosine content; adenine content uracil content.,Section III. Structure and function of RNA,参与蛋白质合成的三种主要 RNA ： messenger RNAs (mRNAs, 5% of total RNA) Transfer RNAs (tRNAs, 15%

21、of total RNA) Ribosomal RNAs (rRNAs, 80% of total RNA),I. Structure and function of mRNA,The most heterogeneous in size and stability. Serve as templates of protein synthesis. Unique characteristics(in eukaryote): cap of 5-terminal (7-mGpppNm) tail of 3-terminal(poly A),mRNA structure in prokaryotes

22、,5,3,Start codon AUG,stop codon,Coding region triplet,5 untranslated region,3 untranslated region,AGGA,Shine-Dalgarno(SD) sequence (Ribosomal binding site, RBS),SD sequence (RBS) is a sequence upstream the start code in prokaryotic mRNA that can base-pairs to a UCCU sequence at or very near the 3 en

23、d of 16S rRNA, thereby binding the mRNA and small ribosomal subunit to each other.,mRNA structure in eukaryotes,5 m7Gppp Cap,AAAAAA 3 Poly A tail,Start codon AUG,stop codon,Coding region triplet,Cap Recognized and bound by CBP (cap-site binding protein) during initiation of translation Poly A tail r

24、ecognized and bound by PAB(poly A binding protein),5 untranslated region,3 untranslated region,. CTCCTAGTACGATCGT.CTAGTCATTCGGTADNA 5cap.GAGGAUCAUGCUAGCA.GAUCAGUAAGCCAU.AAAAA.mRNA N-aa1 aa2 aa3 aa aa-C,5UTR Coding region 3UTR 3polyA tail,capping enzyme,5， 5-GpppG-,AATAAA,TTATTT,GU,GT,CA,DNA,mRNA,7-m

25、GpppNm can bind with CBPs,Functions of 5cap structure ( 7-mGpppNm):,Functions of 3tail (poly A):,poly A bind with PABP,CBP: cap binding protein，帽结合蛋白 PABP:poly(A)-binding protein,多聚腺苷酸结合蛋白,-translocation of mRNA from nuclear to cytoplasm -regulating the initiation of translation -increasing the stab

26、ility of mRNA,Table for genetic codon,hnRNA Splicing,The role of messenger RNAs(mRNA): as template *This class of RNA serves as the templates used to determine the sequence of amino acids in protein synthesis. *In a mRNA, beginning from the first AUG that is said to be the initiation signal(as a ini

27、tiation condon) every subsequent triplet bases is read as a condon until reaching a stop condon (UAA, UGA, UAG). Open reading frame (ORF) 5 C G G A A U G G C A G A G U G G C U A U A A G C A U G Met Ala Glu Trp Leu *Each codon specifies an amino acid.It is the codon sequence of a mRNA that determines

28、 the amino acid sequence of a protein.,Vary in length from 74 to 95 nucleotides. Contains more rare bases. Serve as carrier of aa in the translation.,II . Structure and function of tRNA,rare bases,(C-C-glycosidic bond),rare bases,Features of secondary structure of tRNA : cloverleaf,Four loops: D loo

29、p, anticodon loop, variable loop, and TC loop Four arms: acceptor arm, D arm, TC arm, and anticodon arm,D,D,D,Secondary structure (cloverleaf),Tertiary structure (inverse “L”),D,D,D,The roles of transfer RNAs (tRNAs): as an adapter tRNAs function as an adapter molecule in virtue of forming covalent

30、attachment of individual amino acid catalyzed by aminoacyl-tRNA synthetase and recognizing the codon sequences of the mRNAs to allow correct insertion of amino acids into the elongating polypeptide chain.,Components of ribosomes,III. Structure and function of rRNA,真核生物18S rRNA,原核生物翻译过程中核糖体结构模式:,氨基酰位

31、 （aminoacyl site，A 位）,肽酰位 （peptidyl site，P 位）,排出位（exit site，E 位）,The three roles of RNA in protein synthesis. Messenger RNA (mRNA) is translated into protein by the joint action of transfer RNA (tRNA) and the ribosome, which is composed of numerous proteins and two major ribosomal RNA (rRNA) molecul

32、es,. Other non-coding RNA (ncRNA) long non-coding RNA and small non-coding RNA SnRNA(small nuclear RNA) SnoRNA (small nucleolar RNA) ScRNA(small cytoplasm RNA) siRNA(small interfering RNA) MicroRNAs Small catalytic RNA: Ribozymes, are the RNA molecules with catalytic activity. The activity of these

33、ribozymes often involves the cleavage of a nucleic acid.,RNA interference(RNAi) 被Science评为2002年最重大科技突破。,Small interfering RNA (siRNA),siRNAs structure: a short (usually 21-nt) double-strand of RNA (dsRNA) with 2-nt 3 overhangs on either end.,Ribozyme An RNA molecule with catalytic activity,Secondary

34、 structure of ribozyme - Hammerhead structure,Section Physical and chemical features of nucleic acid and applications,. General features of nucleic acid: Acidity Viscosity Conformation Absorbance of 260nm,1. DNA或RNA的定量 A260=1.0相当于 50g/ml双链DNA 40g/ml单链DNA（或RNA） 20g/ml寡核苷酸 2.判断核酸样品的纯度 DNA纯品: A260/A280

35、 = 1.8 RNA纯品: A260/A280 = 2.0,A260的应用,目 录,Definition: DNA denaturation means that a DNA has lost its native conformation and double strand DNA is separated to single strand DNA by exposed to a destabilizing factor such as heat, acid, alkali,urea or amide. (when high temperature is used to denature DNA, the DNA is said to be melted).,. Denaturation of DNA,DNA melting curve:,Tm is melting temperature at which half (50%) of DNA molecule