1、Chapter 2 Structure and Properties of Nucleic Acid Shihua Wang Ph.D. Prof.,Molecular Biology,1 Overview,1.1.1 DNA Deoxyribose Nucleic AcidFunction: The genetic information carrier, responsible for the genetic information storage and transmissiondistribution： Eukaryotes: nucleus（98%）,organelle（2%） Pr
2、okaryotes: nucleoid、plasmids,1.1 Type and distribution of nucleic acid,1.1.2 RNA,Ribose Ncleic Acid Function: mainly participate the expression of genetic informationType: rRNA（ribosomal RNA）mRNA（messenger RNA）tRNA（transfer RNA）Distribution: Cytoplasm(90%), Nucleus(10%),viruses,mRNA,rRNA,tRNA,1.1.2.
3、1 mRNA,Life time is shortEach polypeptide has corresponding mRNA，the difference among different mRNA is quite big (in length and molecular weight)；Functions: as intermediaries, carrying genetic information from genes to ribosomes, where the corresponding proteins can be synthesized (as templates).,1
4、.1.2.2 tRNA,Molecular weight is the smallestFunctions: as adapter molecules (transport amino acids), that faithfully translate the information in mRNA into a specific sequence of amino acidsEvery Aa at least has a correspnding tRNA（tRNA、tRNAAla）。 Different tRNA has almost the same big，73-98 Nts,1.1.
5、2.3 rRNA,The amount is the most，Molecular Weight is the biggest；Functions: structural components of ribosomes, the complexes that carry out the synthesis of proteins,nucleic acid,nucleotide,1.2 the components of nucleic acid,means pentose, DNA has deoxyribose, RNA has ribose；,means nitrogenous bases
6、,means phosphate,nucleotide,1.2.1 Pentose,ribose （in RNA）,2- deoxyribose （in DNA）,1.2.2 base,DNA,RNA,Modified bases,The chemical modification of bases is widespread, and has a number of specific roles.Such as: the methylation of the N-6 position of A、the 4-amino group and the 5-position of C,1.2.3 n
7、ucleoside and nucleotide,DNA: 2-deoxyribose + A、T、C、G RNA: ribose + A、U、C、GGlycosidic bond: Linking C1 of the ribose to the N1 of pyrimidine Linking C1 of 2-deoxyribose to the N9 of purine,H2O,H2O,base,phosphoric acid,pentose,glycosidic bond,Phosphate bond,1.2.3.2 nucleotide,Nucleoside=base+pentose
8、Nucleotide=base+pentose+phosphate,2 Nucleic acid structure,2.1 primary structure of nucleic aciddefinition：the nucletide residue sequence of the polynucleotide chain； linkage：3,5-phosphodiester bond； backbone：phosphate + pentose； direction： 5 3 ；,Representation,2.2 secondary structure of DNA -DNA do
9、uble helix,Xray photograph of DNA with high quality：DNA specimens from different species have the same results（constant width; 3.4nm）； Chargaff rules：the rule of the composition of DNA Physical chemistry studies and acid and alkali titrate studies on DNA base ；,Experimental basis,Chargaff rules：,The
10、 base composition of DNA varies from one species to another, while DNA specimens isolated from different tissues of the same species have the same base composition； In nature DNA, the number of G units equals the number of C units and the number of A units equals the number of T units ( this strongl
11、y hinted towards the base pair make up of DNA )； The base composition of DNA in a given species does not change with an organisms age, nutritional state,or changing environment。,Chargaff rules：,A Structure for Deoxyribose Nucleic Acid was published by James D. Watson and Francis Crick in the scienti
12、fic journal Nature in its 171th volume on page 737-738 (dated April 25, 1953.) watson-crick.pdf It was the first publication which described the discovery of the double helix structure of DNA.,Double helix stairway in the Vatican Museum,DNA double-helix,Two polynucleotide chains in a DNA double heli
13、xantiparallel（one strand runs in the 53 direction, whlie its partner runs 35）,2.2.1 key notes of DNA double helix,Along the same axis，two chains are wound around each other, resulting in a right-handed double helixforms a major groove and a minor groove,The bases lie on the inside，the sugar-phosphat
14、e backbone is on the outsideThe bases are flat structure, lying in pairs perpendicular to the axis,The diameter of the double helix is 2nmThere is a complete turn every 3.4nm, with 10bp per turn.,Two chains in the double helix associate by hydrogen bonding between the bases, G with C and A with T (
15、base pairing , complementary).Three hydrogen bond can form specifically between G and C, but only two can form between A and T;,2.2.2 Important Meanings,Explains for the first time how genetic information is encoded in DNA and passed from one generation to the nextTremendous strides in our understan
16、ding of the structure of DNA and, as a result our ability to work with and manipulate the information-rich DNA moleculeBase pairing DNA semi-conservative replication mechanism,2.2.3 Stable factors of the double helix,In physiological conditions,the DNA double helix is very stable. stable factors： H
17、bonding between complementary base pairs； Base-stacking interaction（hydrophobic effect, the major factor）； electrovalent bond（between the negative charges carried on the phosphate groups and the positive charges carried on the proteins or metal ions）,2.2.4 DNA can occur in different three-dimendiona
18、l forms,Base Obliquity,helix rise per base pair,bp number per turn,Helical sense,diameter （nm）,B-form,Z-form,0-1 19-20 9,0.34 nm 0.23 nm 0.38 nm,10 11 12,R handed R handed L handed,2.0-2.37 2.55 1.8-1.84,A-form,B-form：relative humidity is 92% A-form：relative devoid of water (under 75%) Z-form：left h
19、anded helix H-form：triple helix,2.3 Structure of RNA,Pentose is ribose，T is replaced by U Most RNA is single strand AT、G C Hairpin or internal loop or bulge；,2.3.1 Primary structure of mRNA,CistronGene。,polyA：20250 A。 Cap：Methyled G，5, 5-linkage。,Cap structure,G Methylation,OCH3,H,Base,Secondary str
20、ucture of tRNA :cloverleafTertiary structure of tRNA: L shape,2.3.2 Secondary and tertiary structure of tRNA,1,2,3,Four stems,Modified bases,cloverleaf,Secondary structure of tRNA,4,Four loops,Aa acceptor arm 3-end and 5-end 3-end is CCA Function：carry actived AaAnticodon arm Have three base called
21、anticodon Function：Match to the code in mRNA，to determine the Aa position,D arm Loop：8-12 Nt Arm：3-4bp Function：aa-tRNA synthesisase binding siteTC arm Have TC Function：binding to ribosomeVariable loop Vary among different tRNA,Tertiary structure of tRNA,L shape,3 Properties of nucleic acid,3.1 gene
22、ral properties of nucleic acidColour：DNA is white fibre，RNA is white powderSolubility：can solve in water，not in organic solvent；,Effect of acid to nucleic acid,Strong acid + high temperature completely hydrolyzed to bases, riboses/deoxyribose, and phosphateModerate acid (PH34) apurinic nucleic acids
23、 glycosylic bonds attaching purine (A and G) bases to the ribose ring are broken ,Effect of alkali to nucleic acid,High pH denatures DNA and RNA by altering the tautomeric state（互变异构态）of the bases and disrupting specific hydrogen bonding. RNA is also susceptible to hydrolysis at high pH, by particip
24、ation of the 2-OH in intramolecular cleavage of the phosphodiester backbone.,DNA denaturation at high pH,keto form（酮式）,enolate form （烯醇式）,keto form,enolate form,Base pairing is not stable anymore because of the change of tautomeric states of the bases, resulting in DNA denaturation,RNA hydrolyzes at
25、 higher pH because of 2-OH groups in RNA,RNA is unstable at higher pH,OH,free 5-OH,2, 3-cyclic phosphodiester,alkali,Viscosity,DNA solutions have a high viscosity, while RNA decreases sharply,Reasons for the DNA high viscosity High axial ratio, because DNA is very long and thin Relatively stiff,Appl
26、ications: Long DNA molecules can easily be shortened by shearing force. When isolating very large DNA molecule, always avoid shearing problem,Buoyant density,The density of DNA is typically slightly greater than 1.7 g/cm3, while the density of RNA is more than 1.8 g/cm3. Proteins have densities less
27、 than 1.3 g/cm3,RNA pellets at the bottom,Protein floats,Purifications of DNA: equilibrium density gradient centrifugation,1.6600.098GC,3.2 nucleic acid enzymolysis,According to substrates：DNases、RNasesAccording to function methods：exonucleases、endonucleases,exonucleases：catalyze the hydrolysis of s
28、ingle nucleotides from the end of a DNA or RNA chain；,Endonucleases：catalyze the hydrolysis of bonds between nucleic acids in the interior of DNA or RNA sequence.Restricition endonuclease recognizes specific DNA sequences and cut the DNA backbone at a specific site within that sequence,3.3 UV absorp
29、tion properties,nucleic acids absorb UV light due to the aromatic bases max=260nmApplication: Detection Quantitation Assessment of purity,assessment of purity RNA： OD260 / OD280=2.0DNA ：OD260 / OD280=1.8Protein: OD260 / OD280=0.5,quantitation 1 mg/ml dsDNA has an A260 of 20ssDNA and RNA 25 OD260 = 1
30、.0 = 50g/mL dsDNA= 40g/mL ssDNA/RNA= 20g/mL (d)NTP,3.4 Denaturation,renaturation and hybridization,3.4.1 Denaturation 3.4.1.1 Definition: a number of physical and chemical factors can lead to the destruction of double-stranded hydrogen-bonded regions of DNA and RNA. the double-stranded nucleic acids
31、 are converted to single strands.,Denaturation of circular DNA: If both strands are closed circles, denaturation disrupts the double helix, but the two single strands become tangled about each other and cannot separate.(b) If one or both strands are nicked, however, the two strands will separate on
32、thermal denaturation.,3.4.1.2 Denaturation factor,pH（11.3或5.0） Chemical denaturation (urea、formaldehyde) Thermal denaturation Low ion strength,3.4.1.3 Characters of denatural DNA： biological activity changed (even lost)； hyperchromicity,viscosity decreased，solubility decreased, buoyant density incre
33、ased； The structure became loose, easy to be hydrolyzed； Molecular weight remained unchanged,The absorbance of isolated nucleotides is greater than that of RNA and ssDNA, which is in turn greater than that of dsDNA.,Hypochromicity,Hypochromicity dsDNA is hypochromic with respect to ssDNA Hyperchromi
34、city ssDNA is hyperchromic with respect to dsDNA.,3.4.1.4 thermal denaturation and Tm,Increased temperature can bring about DNA denaturation; Tm （melting temperature): Temperature when 50% DNA denaturation Tm is a characteristic constant of DNA,RNA: the absorbance increases gradually and irregularly
35、 DNA: the absorbance increases cooperatively.,Effection factor to Tm：,G+C amount： G+C is high，Tm is high。（G+C）%=（Tm-69.3）2.44,DNA uniformity：If uniformity is good，Tm range is small,Ion strength：DNA should save in buffer with salt,3.4.2 Renaturation,3.4.2.1 Definition：annealing。,Anneal,3.4.2.2 Charac
36、ters of renaturation biological activity resumed hypochromicity viscosity increased, solubility increased, buoyant density decreased,3.4.2.3 Factors influencing the renaturation,temperature：slowly decrease DNA concentration: high is good Size of DNA segments：fragment small is good Buffer composition
37、 (mostly salt concentration) DNA complexity： repetitive segment the more is good,3.4.3 hybridization,Definition：the renaturation of regions of complementarity between different nucleic acid strands(DNA or RNA) Characteristic：sensitive、specific,DNA-DNA hybridization,Different DNA,Southern Blotting,No
38、rthern Blotting,4.1 Supercoiling:Supercoiling is the coiling of the DNA axis upon itself。,4. DNA supercoiling,Positive supercoils： （if the strands are overwound）Negative supercoils： （if the strands are underwound）,Positive supercoils,Negative supercoils,DNA isolated from cell negatively supercoiled
39、by 5 turns per 100 turns of the helix. Lk / Lk = -0.05,Lk = Lko,Relaxed covalent closed circular,4.2 Topoisomer,Topoisomer : A circular dsDNA molecule with a specific linking number which may not be changed without first breaking one or both strands.,链环数（linking number）： 表示超螺旋的拓扑性质。 一个封闭环状dsDNA分子两条链
40、彼此交叉的次数 L=T+W,T(Twist): 表示双螺旋的螺圈数； T总碱基对/每一螺圈的碱基 W(writhe): 表示超螺旋的程度， W双螺旋中心轴盘绕的圈数。,Topoisomerases: exist in cell to regulate the level of supercoiling of DNA molecules Type I topoisomerase: break one strand of the DNA , and change the linking number in steps of 1 by passing the other strand through
41、 the break. Type II topoisomerase: break both strands of the DNA , and change the linking number in steps of 2 by transferring the other dsDNA through the break.,4.3 Topoisomerases,Type I topoisomerase,Type II topoisomerase,intercalator (EB） locally unwinding of bound DNA, resulting in a reduction in twist and increase in writhe.,4.4 intercalator,学 习 要 求,掌握核酸的种类、分布及功能； 掌握核酸的分子组成，连接方式，表示符号； 掌握DNA二级结构的内容（双螺旋要点，意义，稳定因素，碱基配对原则等）； 掌握tRNA二级结构的内容； 熟悉核酸的颜色反应； 掌握核酸的紫外吸收，变性、复性，分子杂交；,
