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1Chapter IV Nucleic Acid Structure and Function2 (I)Secondary StructureDouble helix Dr.Crick Dr.WatsonI.DNA StructureX-Ray Diffraction Photograph of a Hydrated DNA Fiber by Dr.Maurice Wilkins3The central cross is diagnostic of a helical structure.The strong arcs on the meridian arise from the stack of nucleotide bases,which are 3.4 apart.1.The base composition of DNA generally varies from one species to another.2.DNA specimens isolated from different tissues of the same species have the same base composition.3.The base composition of DNA in a given species does not change with an organisms age,nutritional state,or changing environment.4.In all cellular DNAs,regardless of the species:A=T，G=C，A+G=T+C4 Chargaff rules:56 Features of double helix(1)：1.Two antiparallel helical DNA chains wound around the same axis to form a right handed double helix，diameter is 2nm。2.The hydrophilic backbones of alternating deoxyribose and phosphate groups are on the outside of the double helix,facing the surrounding water.3.The purine and pyrimidine bases of both strands are stacked inside the double helix,with their hydrophobic and nearly planar ring structures very close together and perpendicular to the long axis.78Features(2):4.The pairing of the two strands creates a major groove and minor groove on the surface of the duplex.5.10 base pairs,or 34 (3.4 nm),per turn of the helix.6.The hydrogen-bonded base pairs:A:T 2 Hydrogen bonds G:C 3 Hydrogen bonds9Hydrogen bonds between bases join the 2 strands.10Features(3):7.The double helix is stabilized by hydrogen bonds and base stacking force(hydrophobic interaction and van der Waals forces）Unstable factors：electrostatic repulsion between negative charged phosphate groups11B form DNA：the most stable structure for a random-sequence DNA molecule under physiological conditions.12Features：left hand helix，zigzag appearance，only has a narrow and deep minor groove pyrimidines alternate with purines may play a role(as yet undefined)in regulating gene expression or in genetic recombination.Z form DNA(Zigzag DNA)13The triplexes are most stable at low pH.The triplexes also form most readily within long sequences containing only pyrimidines or only purines in a given strand.14Triplex DNA15(II)Tertiary Structure of DNASupercoil 超螺旋超螺旋The DNA in a prokaryotic cell is a supercoil.Negative SupercoilOpen circular,relax DNA16nElectron Microscope Diagram of DNA SupercoilElectron Micrograph of Part of the E.coli genomeNucleosome（核小体）：（核小体）：the first and most basic level of chromosome packing1.nucleosome core：histone octamer （2 molecule each of H2A,H2B,H3,H4）1.75 turn of DNA supercoil （146bp）2.linker DNA：histone H1,DNA double helix (20-80bp)17(III)Quaternary Structurecombined with protein Eukaryote：Chromosome(染色体染色体)1819 DNA chromatin looks like a string of beads.nElectron microscope diagram of DNA chromatin20DNA is tightly packed with an overall compaction ratio of approximately 8000-10000-fold to 2 m in length(fit inside a cell).Nucleosome（核小体）（核小体）30nm fiber（纤丝）（纤丝）150nm Radial loops（突环）（突环）300nm Rosette（玫瑰花结）（玫瑰花结）700nm Coid（螺旋圈）（螺旋圈）1400nm Chromosome（染色体）（染色体）nuclear scaffold30nm fiber21 II.Gene and Genome（I)Gene(基因基因):A segment of nucleic acid code for a complete particular protein or synthesize a RNA.Gene is the functional unit of nucleic acid.22（II）Genome（基因组（基因组）：the total genes and intergenic sequences in a living cell or living beings.Store all genetic information of one species.231.Virus genome:Only one kind of nucleic acid Overlapping gene242.Prokaryote Genome：l Circular double strand DNAl Most of genes are structural genesl Single copy genel Operon(操纵子）操纵子）regulate gene expression25DNAmRNAproteintranscriptiontranslationABPolycistronRegulatory sequenceoperon261.Split genes(分割开的基因分割开的基因)：Intron(内含子内含子)，Exon（外显子）外显子）2.Unique sequence（单拷贝序列（单拷贝序列 )3.Moderately Repetitive Sequence（中度重复序列）（中度重复序列）4.Highly Repetitive Sequence(高度重复序列高度重复序列)3.Eukaryote Genome2728l Highly repetitive fraction consists largely of satellite DNA,simple sequences repeated 105 times per haploid genome.l Extreme G+C contents,short units l Most satellite DNA is located at centromeres and telomeres Fragmented DNA was centrifuged to equilibrium in a cesium chloride gradient.Most of the genomic DNA migrates as a main band at a density of 1.7.Satellite DNA migrates at a lower density.29III.RNA Structure30regional double helixloopBase pairing stemhairpin（stem-loop）No base pairing loop31Spatial StructureRNADNASecondarySecondaryirregularirregular，commonly in commonly in hairpin structurehairpin structuredouble helixdouble helixTertiaryTertiarydiversitydiversitysupercoilsupercoilQuaternaryQuaternaryinteraction with proteininteraction with proteinIntra-strand pairingInterstrand pairing32(I)tRNAtRNAs compose roughly 20%of total cellular RNA.Function：Transport amino acids to ribosomes for assembly into proteins.1 tRNA1 tRNA1 amino acid1 amino acid1 amino acid1 amino acid1 tRNA1 tRNAtRNAAmino acidaminoacyl tRNA synthetaseAminoacyl tRNAAminoacyltRNAAminoacyl tRNA mRNA53331.Primary Structure of tRNA：7393 nucleotides single strand RNA 30%of bases is invariable related to fundamental function.Some variable bases recognition site of aminoacyl tRNA synthetase6HNNNHON6deamination5566（U）（DHU）hydrogenation（A）（I）5end:pG 3end:-CCA-OH post-transcription modification 10%rare base and nucleoside dihydrouracil（DHU）、）、hypoxanthine（I）、）、pseudouridine（）、）、ribothymine（rT）。）。34Rare NucleosideOOHOHHOCH2HNOON234561OOHOHHOCH2HNOONH125346UridinePseudouridine（psai：）：）352.Secondary Structure of tRNA warped cloverleaf with four loops and four arms Amino acid arm(7bp):to bind amino acid D loop(8-14bp)and D arm(3-4bp):contain DHU Anticodon loop(5bp)and arm(7bp):to recognize genetic code in mRNA TC loop(7bp)and arm(5bp)Variable loop(4-5bp or 13-21bp):main taxonomic features 36Codon：5 GCC 3Anticodon：5 CGG 3Antiparallel pairingAdenineAminoacyl tRNA373.Tertiary Structure of tRNA-L shaped38（II）mRNA（信使（信使RNA）Function：template in protein synthesis。391.Prokaryote mRNA Structure(1)Polycistron：several structural genes transcript to one mRNA.(2)Intergenic sequences in the middle and untranslated sequences at two ends.Protein AProtein BProtein C5 ppp3 OH5noncoding sequence3noncoding sequenceIntergenic sequence40(3)ShineDalgarno sequence（SD）AUGAGGA53Initiation codonSDsequence712ntmRNAUCCU16rRNA 35PARibosomeSD sequence of ProkaryoteA segment of conserved sequence rich in purines at the upstream of 5 AUG of prokaryote mRNA.Complemented with ACCUCCU at 3 end of 16S rRNAThe segment is related to mRNA recognition and binding in translation.41（4）Need not modification after transcription,directly translate to protein.DNAmRNAProteintranscriptiontranslation53N(5)Half life is short42 2.Eucaryote mRNA structure：（1）Monocistron:one structural gene transcript to one mRNA.AUG coding sequence UGAm7G5ppp5Nm(Nm)AAUAAAAAAA(n)OHm6Am6A5noncoding sequence3noncoding sequencepolyA site43(2)Heterogeneous nuclear RNA(2)Heterogeneous nuclear RNA （hnRNAhnRNA）is is modified after transcription.modified after transcription.细胞质细胞质细胞核细胞核DNA内含子内含子外显子外显子转录转录转录后剪接转录后剪接转运转运成熟成熟mRNAhnRNA翻译翻译蛋白质蛋白质44The 5 cap(red)is added before synthesis of the primary transcriptis complete.A noncoding sequence following the last exon is shown in orange.Splicing can occur either before or after the cleavage and polyadenylation steps.All the processes shown here take place within the nucleus.Maturation of mRNA in a eukaryotic cell.(3)5cap structure7-methyl guanosine55phosphate triester bond2OH methylationm7GpppN m7GpppGmpN m7GpppGmpNm helps stabilize the mRNA by preventing the attack of 5exonucleases involved in the recognition of mRNA by the translation machinery4546(4)3polyA tailpolymer of adenylate residues 20250 nucleotides in the 3-terminal of mRNAAAAAAAAAA53add post-transcriptionallyAAAAAAAAATTTTTTTTTTDNA（）（）（）（）5533mRNA？47nThe major steps in generating the 3 end48exception：histoneapplication：extraction of mRNAoligoT/UtRNA、rRNAmRNATotal RNAreverse transcription of mRNAAAAAAATTTTTTToligoT primermRMAcDNA5533Reversetranscriptasemaintains the intracellular stability of mRNA 49Kozak sequence：CCAUGGGCCAG-3+4Initial codonmRNA 53A segment of conserved sequence around 5AUG of eukaryote mRNA.Related to accuracy and efficiency of translation initiation.Eukaryote Kozak sequence50(6)Modified base in mRNAProkaryote：NoEukaryote：m6AAdenine66-methyl-adenineCH36methylation(5)Half life of eukaryote mRNA is longer51Prokaryote and Eukaryote mRNA structureProkaryoteEukaryotepolycistronmonocistronSD sequenceKozak sequenceno5 capno3 tailT1/2 shortT1/2 longNo rare baseHas rare base52(III)rRNA（核糖体核糖体RNA）53Prokaryote(E.Coli)Eukaryote(mouse liver)Small Subunit30S40SrRNA16S1542 nucleotides18S1874 nucleotidesProtein21kinds40%of total weight33kinds50%of total weightLarge subunit50S60SrRNA23S5S2904 nucleotides120 nucleotides28S5.8S5S4718 nucleotides158 nucleotides121 nucleotidesProtein34kinds30%of total weight49kinds35%of total weight54CGAAC5556(IV)Small RNA1.snRNA（细胞核内小分子（细胞核内小分子RNA）2.snoRNA（核仁小（核仁小RNA）3.scRNA（胞质小分子（胞质小分子RNA)4.Antisense RNA（反义（反义RNA）5.Ribozyme(核酶核酶)6.siRNA（小干扰（小干扰RNA）7.miRNA（微（微RNA）571.snRNA（small nuclear RNA）Types：U1、U2、U4、U5、U6 （snRNA pr snRNP）Function：involved in rRNA and mRNA processing 5353AGGUU1U5hnRNAU1U55hnRNA35mRNA3Exon2Intron1Exon1582.Small nucleolar RNA（snoRNA,小核仁小核仁RNA）Function：participate in assembly of ribosome and processing3.Small cytosol RNA RNA（scRNA）7SL RNA7SL RNAPrSignal Recognition Particle（SRP）SRPSRP receptorEndoplasmicReticulumNCSignal Peptide594.Antisense RNA a single-stranded RNA that is complementary to a mRNA strand transcribed within a cell。When mRNA forms a duplex with a complementary antisense RNA sequence,translation is blocked.mRNAAntisense RNAProteinHybridizationTranslation605.Ribozyme RNA molecules that have catalytic activity.Dr.Thomas R.CechDr.Sidney Altman In 1989,Thomas R.Cech and Sidney Altman won the Nobel Prize in chemistry for their“discovery of catalytic properties of RNA.”616.siRNA6.siRNA (小干扰小干扰RNA)RNA)RNA interference(RNAi)is a biological process in which RNA molecules inhibit gene expression,typically by causing the destruction of specific mRNA molecules.Historically,it was known by post transcriptional gene silencing(PTGS).Two types of small RNA microRNA(miRNA)and small interfering RNA(siRNA)are central to RNA interference.RNA interference has an important role in defending cells against parasitic nucleotide,but also in directing development as well as gene expression in general.62for their discovery of RNA interference gene silencing by double-stranded RNAThe Nobel Prize in Physiology or Medicine 2006 Andrew Z.Fire Craig C.Mello Potent and specific genetic interference bydouble-stranded RNA in Caenorhabditis elegansNature,199863(1)Self DefenseDefense against virus and exogenous DNA invasionFunction：(2)Used as a gene knockdown tool64RNA干扰机制干扰机制1、启动阶段：、启动阶段：dsRNA被被Dicer酶逐步切割成酶逐步切割成siRNA（short interfering RNA）。2、效应阶段、效应阶段:siRNA与酶聚集形成与酶聚集形成RNA诱导沉默复合物诱导沉默复合物Risc（RNA-induced silencing complex）。）。Risc的激活：的激活：siRNA双链解开。双链解开。激活的激活的Risc定位到同源定位到同源mRNA上，降解该上，降解该mRNA。DicerRNase III家族成员核酸内切酶，特异性识别并切割家族成员核酸内切酶，特异性识别并切割dsRNA65mechanism of RNAiRISC：RNA-induced silencing complex（RISC）Genome has genes that code for microRNAs Regulate 30%of mammalian gene products Have been described in invertebrates and vertebrates:worms,fungi,plants,and mammals Many are conserved between vertebrates and invertebrates7.microRNA(miRNA）Definiton：a noncoding RNA with stem-loop structure,typically 20-24nucleotides in length.Paired with target mRNA,arise mRNA cleavage or translational repression.66Esquela-Kerscher,A.,and Slack,F.J.(2006).Nature Reviews 6,p.263-Primary-miRNA is transcribed in the nucleus,and is usually several kilobases long;posses 5 cap and a poly-A tail.-Cleaved in the nucleus by Drocha enzyme to 70nt hairpin transcript(pre-miRNA).-Transported to the cytoplasm by Exportin 5 through nuclear pores.-Cleaved by Dicer into 20-24nt ds-transcripts.-one strand miRNA is incorporated into the RNA-induced silencing complex(RISC)-paired with target mRNA and lead to mRNA cleavage or translational repression。67 siRNA miRNA6869(I)General Properties1.Macromolecule：Mammals 3109bp Unit of Nucleic acid size（1）Dalton（2）base pair（bp）（3）chain length（m）（4）sedimentation coefficient（S）3000bp=2106 Dal=1 m Methods to analyze the molecule size：Gel Electrophoresis，Ultracentrifugation，SequencingIV.Physiologic chemical Properties 70Sanger Sequencing:key reagent:dideoxynucleotides triphosphates(ddNTP)NNNNNH2OOPOO-OPOO-OPO-OO-NHNNNOOOPOO-OPOO-OPO-OO-ddATPNH2ddGTPOOPOO-OPOO-OPO-OO-OOPOO-OPOO-OPO-OO-ddTTPddCTPNNHOONNNH2OWhen a ddNTPis incorporatedelongation of the primer is terminatedThe ddNTP isspecificallyincorporatedopposite itscomplementarynucleotide base DNA polymerase35-32P35primerTemplateunknown sequenceMg2+,dNTPs+one ddATPON3HONNHOOOHONNNNHOSOOHNNH2NOOHONNNH2OddIAZT(-)-3TCAnti-Viral Nucleosidesd4C71Sanger SequencingLarger fragmentsSmallerfragmentsGTAACGTAATCACAGddAddGddCddTCATTGCATTAGTGTC532P35332P-53primertemplate72Fluorescence-based dideoxy-DNA sequencing737475 endonuclease(核酸内切酶核酸内切酶)：tool enzyme exonuclease(核酸外切酶核酸外切酶):part of DNA replication5 GAATTC3 5G3 5AATTC33 C TTAAG5 3 CTTAA5 3 G5 2.Hydrolysis of nucleic acidEcoRI7677(II)Denaturation and Renaturation 1.denaturation(变性变性)Definition：double strands single strandThe double-stranded structure of DNA is separated into two single component strands when hydrogen bonds between the paired bases are disrupted under some physical and chemical conditions.78 Hyperchromic Effect(增色效应）：增色效应）：When nucleic acid is denatured,the inner bases are exposed,thereby the value of 260nm absorption is increased.1.Nature DNA2.Denatured DNA3.Total UV absoption of nucleotides79 Melting Temperature（解链温度（解链温度,Tm）When DNA is denatured,the temperature at which UV absorption increase to an half of maximum value is defined as Tm.85-95G-C content and ionic strength are in direct proportion to Tm.80Low Na+High Na+81 Impact factors:DNA concentration DNA length DNA complexity Temperature =Tm-25 Ionic Strength：0.4mol/L2.renaturation(复性复性)or Annealing（退火）（退火）Definition:When slowly cooling down the denatured DNA solution,the single strand DNA can reform a double strands helix to recover its biological functions.8283
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