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1、Chapter 8 Nucleotides and nucleic acids History of the research Nucleic acid structure Nucleic acid chemistry Other functions of nucleotides2012-10-30 Proteins, carbohydrates, lipids, nucleic acids are the four categories of macromolecules, necessary for life. Nucleic acids play a critical role in t
2、he storage and function of genetic information in all living organisms.“ A genetic material must carry out two jobs: duplicate itself and control the development of the rest of the cell in a specific way.” -Francis Crick Overview 1869F. Miescher從膿細(xì)胞核中提出含磷量高的核 素（nuclein），其后從鮭魚精子中提取出 魚精蛋白和核素。 1885O. H
3、ertwig提出核素可能負(fù)責(zé)受精和遺傳。 1889R. Altmann從酵母和動(dòng)物組織中提取出不含 蛋白質(zhì)的核酸。 1894A. Kossel和A. Neumann從胸腺中提取核酸， 表明胸腺核酸與酵母核酸不同。 O. Hammars證明酵母核酸中的糖是戊糖。 History( (研究方向：核酸化學(xué)、遺傳學(xué)、結(jié)構(gòu)學(xué)、方法學(xué)研究方向：核酸化學(xué)、遺傳學(xué)、結(jié)構(gòu)學(xué)、方法學(xué)) ) 1902E. Fischer研究糖和嘌呤而獲諾貝爾化學(xué)獎(jiǎng)。 核酸中的嘌呤和嘧啶主要由Kossel等人所鑒定。 1909P. A. Levene和W. A. Jacobs鑒定D-核糖。 1910Kossel因核酸化學(xué)研究中的成就
4、而獲諾貝爾 生理學(xué)獎(jiǎng)。 1912P. A. Levene認(rèn)為核酸由四種核苷酸等量聚 合而成。 1929Levene和Jacobs確定胸腺核酸中的糖是D-2- 脫氧核糖。 1931 Levene在“Nucleic Acid”一書中指出核酸分為 動(dòng)物核酸（zoonucleic acid）和植物核酸 （phytonucleic acid）。 1933J. Brachet發(fā)展了組織化學(xué)的方法。 1938T. Caspersson顯微紫外分光光度技術(shù)。 1943J. N. Davidson用化學(xué)分析方法支持上述組織化學(xué)和紫外分光光度法得出的結(jié)論：動(dòng)、 植物組織均含有兩類核酸。 1946-50E. Cha
5、rgaff測(cè)定了DNA堿基，提出Chargaff 法則。 1944O. T. Avery肺炎雙球菌轉(zhuǎn)化實(shí)驗(yàn)。 1952A. Hershey和M. Chase證明噬菌體感染時(shí)只 有DNA進(jìn)入細(xì)菌細(xì)胞。 1953J. D. Watson和F. H. C. Crick提出DNA雙螺 旋結(jié)構(gòu)模型。1962年與M. H. F. Wilkins共獲 諾貝爾生理學(xué)獎(jiǎng)。1969年噬菌體小組的M. Delbrk、A. D. Hershey和S. E. Luria也獲諾 貝爾獎(jiǎng)。 1958M. Meselson和F. W. Stahl用超速離心同位素 標(biāo)記的DNA，證明了DNA的雙螺旋結(jié)構(gòu)。 F. H. C. C
6、rick提出中心法則。 1959S. Ochoa和A. Kornberg由于酶促合成RNA 和DNA而獲諾貝爾獎(jiǎng)。 196061 F. Jacob和J. Monod發(fā)現(xiàn)mRNA并提出操縱 子學(xué)說，1965年與P. Rous一起獲諾貝爾獎(jiǎng)。 1965M. W. Nirenberg等破譯了遺傳密碼。1968年 R. W. Holley、H. G. Khorana和M. W. Nirenburg共獲諾貝爾獎(jiǎng)。 1970H. Temin和D. Baltimore發(fā)現(xiàn)逆轉(zhuǎn)錄病毒。 1970 H. O. Smith和K. W. Wilcox發(fā)現(xiàn)類型II限制性 內(nèi)切酶。 1971 K. Danna和D. N
7、athans作出SV40 DNA的限制性圖 譜。1978年諾貝爾化學(xué)獎(jiǎng)授予發(fā)現(xiàn)限制酶的 W. Arber、 H. O. Smith和D. Nathans。 1972 P. Berg與其工作者將SV40 DNA與噬菌體 DNA重組。 1973 S. Cohen等獲得第一個(gè)DNA重組體克隆。 1975 F. Sanger和A. R. Coulson發(fā)明酶法DNA快速測(cè)序。 1977 A. M. Maxam和W. Gilbert發(fā)明化學(xué)法DNA測(cè)序。 1980年Berg、Gilbert和Sanger共獲諾貝爾化學(xué)獎(jiǎng)。 1985 K. Mullis發(fā)明PCR技術(shù)。1993年與發(fā)明定位誘變的 M. Sm
8、ith共獲諾貝爾化學(xué)獎(jiǎng)。Swiss physician Friedrich Miescher (1844-1895) during 1869-1871 Felix Hopper-Seylers lab localted in Tubingens castleThe nuclein isolated from salmon sperm by F. MiescherDiscovery of Nuclein (核素的發(fā)現(xiàn))Friedrich Mieschers interesting findings:Human Genet 2008, 122: 565-581 Aim: determine the
9、 biochemical composition of lymphocytes (later, changed to leukocytes); the novel substance could be precipitated by acidifying and re- dissolved with alkaline solution; The nuclei was separated from cytoplasm, the new substance were obtained from nuclei; Contained a large portion of phosphorous; Re
10、sistant to protease digestion Conclusion : unlike proteins or lipids, it was called nuclein Speculate: store phosphorous or act as a reservoir for other molecules derived from nuclei. Friedrich Mieschers paper(“On the chemical composition of pus cells”)(Medical-Chemical Investigation)Nucleic acid st
11、ructureNucleotidesNucleotide (核苷酸): (1) nitrogen-containing base (2) a pentose (3) a phosphateNucleoside (核苷)RNA: D-ribose DNA: 2-deoxy-D-ribosePyrimidine and Purine bases NomenclatureConformations of riboseequilibriumRibofuranose rings can exist 4 conformations 12345 (A)(G)(C)(T)(U)Bases in DNA: A
12、G C TBases in RNA: A G C UDNARNAMinor purine and pyrimidine bases (nucleosides)Some adenosine monophosphateswere formed by RNA hydrolysisAltered or unusual bases in DNA often have roles in regulating or protecting the genetic information DNA methylation Minor bases of many types are found in RNA, es
13、pecially tRNARNA and DNA are chains of either ribonucleoside monophosphates or deoxyribonucleoside monophosphates covalently joined together by phosphodiester linkages (primary structure). Long chains of nucleotides are called polynucleotides; Short chains of nucleotides are called oligonucleotides.
14、 Phosphodiester linkages in the covalent backbone of DNA and RNA The phophodiester linkages have a direction (called polarity; the convention is 5 to 3, from left to right) The arrangement (or order) of specific nucleotides along the chain is called the sequence containing genetic information. RNA i
15、s hydrolyzed under alkaline condition, but DNA is not. Properties of nucleotide bases weakly basic and aromatic compounds; pyrimidines are planar, and purines are very nearly planar; exist in two or more tautomeric forms depending on pH; hydrophobic stacking interaction which is important in stabili
16、zing the three dimensional structure; hydrogen-bonding is the most important mode of interaction between two complementary strands of DNA. Tautomeric forms of Uracil (互變異構(gòu)式)pH = 7Nucleic acids are characterized by a strong absorption at 260 nmBase pairs: A to T (or U) G to CHydrophobic base stacking
17、 interactions are important in stabilization of nucleic acid structures Hydrogen-bondingNucleic acid structure(In 1968, Watson wrote The Double Helix, one of the Modern Librarys 100 best non-fiction books )Establishment of the structure was based on : i) DNA composition (polydeoxyribonucleotide); ii
18、) X-ray diffraction of DNA fiber; iii) Chargaffs discoveryChargaff (Erwin Chargaff: 1905-1995) rules: The base composition of DNA varies from species to another; DNA specimens isolated from different tissues of the same species have the same base composition; In all cellular DNAs: quantitative relat
19、ionship between the bases (A = T; G = C)X-ray diffraction pattern of DNAThe spots forming a cross in the center denote a helical structure. The heavy bands at the left and right arise from the recurring bases. The data was suggested that DNA molecules are helical with two periodicities along their l
20、ong axis.Watson-Crick model for the structure of DNAIt has not escaped our notice It has not escaped our notice that the specific pairing we have postulated suggests a possible copying mechanism for the genetic material. Watson-Crick Model It consists of two helical DNA chains wound around the same
21、axis to form a right-handed double helix; The hydrophilic backbones of alternating deoxyribose and phosphate groups are on the outside of the double helix; The purine and pyrimidine bases of both strands are stacked inside the double helix; Hydrogen-bonded base pairs: G C ; A=T; Two complementary DN
22、A strands are antiparallel. Helix diameter is 2.0 nanometers. Helix rise per base pair is 0.34 nanometers. Helix pitch (distance along the axis per 360 degree turn) is 3.4 nanometers. Ten base pairs per helical turn. (B-DNA has an average of 10.5 base pairs per helical turn.) The precipitating event
23、sIn 1953, Linus Pauling published his triplex model in PNAS (Vol 34: 84-97)Replication of DNA as suggested by Watson and Crick1928- Watson, Crick, and Wilkins were awarded the 1962 Nobel Prize in Physiology or Medicine for their discoveries concerning the molecular structure of nucleic acids and its
24、 significance for information transfer in living materialHowever, no mention of Franklins contribution !DNA occur in three-dimensional forms conformation of deoxyribose; rotation about the bonds of the phosphodeoxyribose free rotation about the C-1-N-glycosyl bondFree rotation about the C-1-N-glycos
25、yl bond, resulting intwo conformations, syn and anti A, B, and Z forms of DNA (different three dimensional forms)36 base pairComparison of A, B, and Z forms of DNA(occurrence is uncertain)Right hand helixB form DNARight hand helixA form DNAl major groove is deepl minor groove is shallowerLeft hand h
26、elixZ form DNAl minor groove is narrow and deep Base pairs are rotated in Z-DNA Double-stranded RNA and DNA-RNA hybrid duplexes essentially only exhibit the A-form secondary structure. No naturally occurring long DNA molecule has been shown to be entirely in the form of Z-DNA. Regions of naturally o
27、ccurring DNA can form Z-DNA. Regions of alternating purine-pyrimidine (for example, CGCG, etc.) are more prone to form Z-DNA. DNA can assume other secondary structures called A-DNA and Z-DNA The conformation of the base relative to the sugar is important for Z-DNA. Bases are ONLY in the anti conform
28、ation in A-DNA and B-DNA. Pyrimidines usually cannot adopt the syn-conformation. In Z-DNA the alternating CGCG has alternating anti-syn conformations. Formation of a region of Z-DNA in B-DNA unwinds the DNA (a change from a right-handed helix to a left- handed helix).Certain DNA sequences adopt unus
29、ual structuresPalindromes: sequences of double-stranded nucleic acids with twofold symmetry.Minor repeat: a symmetric sequence within each strand.Hairpins and cruciforms structures are formed based on palindromic DNA (RNA) sequencesHairpins and cruciforms structures are formed based on palindromic D
30、NA (RNA) sequencesDNA structures containing three or four DNA strandstriplex DNAHoogsteen pairing: non-Watson-Crick paringN-7O6N6Triple helix DNAGuanosine tetraplex (G tetraplex)Quadruplex DNAPeptide Nucleic Acid (PNA) PNA is a nucleic acid analog in which the sugar phosphate backbone of DNA has bee
31、n replaced by a synthetic peptide backbone usually formed from N-(2-amino-ethyl)-glycine units, resulting in an achiral and uncharged mimic. Peptide Nucleic acid(PNA) Achiral, peptide-like backbone Backbone is uncharged High thermal stability High-specificity hybridization with DNA Resistant to enzy
32、matic degradation It can displace DNA strand of duplex Pyrimidine PNA strands can form 2:1 triplexes with ssDNABiotechnological applicationsRNABacterial mRNAMessenger RNAs (mRNAs) code for polypeptide chainsIn bacteria and archaeaMonocistronic: a single mRNA codes for only one polypeptide Polycistro
33、nic: a single mRNA codes for two or more different polypeptodes.Single-stranded RNA (right -handed)RNA types: mRNA (messenger RNA) tRNA (transfer RNA) rRNA (ribosomal RNA) miRNA (microRNAs) siRNA (small interfering RNAs) Base-stacking interactionSecondary structure of RNAsRNA base paring pattern: G
34、pairs with C, A pairs with UA-form right-handed helix (A-form is predominant)Base-paired helical (secondary) structures in an RNAM1 RNA component of E.Coli RNase PNon-Watson-CrickG=U base pairThree-dimensionalstructure in RNATo understandthe relationshipbetween RNA structureAnd its function !tRNArib
35、ozymean intronDNA denaturation and annealing reversible process temperature or extreme pH affects the process hydrogen bonds are brokenEach DNA species has a a characteristic denaturation temperature (tm, melting temperature)Tm depends on pH and ionic strength and the size and DNA base composition.P
36、artially denatured DNA was visualized by Electron microscopy The arrows point to single-stranded bubbles where DNA denaturation occurred.Formation of hybrid duplexes of DNAIf the two DNAs have significant sequence similarity, they tend to form duplexes or hybrids.Nonenzymatic reactions of nucleotide
37、s AP site (abasic site)N- -glycosyl bondAlteration in DNA structure leads to mutation Cytosine deamination (uracil) occurs in about one per 107 cytidine residues in one day. DNA depurination occurs at a higher rate for purine (one per 105 purines) than for pyrimidines. RNA depurination is much slowe
38、r and not significant. Abasic sites are the most frequently occurring cellular DNA damage (generated spontaneously). Formation of pyrimidine dimers induced by UV lightTwo types: Cyclobutane thymine dimer 6-4 Photoproductcyclobutane thymine dimerChemical agents that cause DNA damage(promote deaminati
39、on)An example of guanine methylation(methylguanine cannot base-pair with cytosine)dimethylsulfateDNA methylation DNA methylation is a biochemical process that is important for normal development in higher organisms. Addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the
40、number 6 nitrogen of the adenine purine ring,is catalyzed by methylase. This modification can be inherited through cell division. Adenosine residues within the sequence 5GATC 3 are methylated. In eukaryotic cells, 5% of cytidine residues are methylated, most common at CpG sequences. DNA methylation
41、at the 5 position of cytosine has the specific effect of reducing gene expression (vertebrate). Common In bacteriaCommon in eukaryoteNucleic acid chemistryNobel Price in Chemistry in 1980Paul Berg( 1926- )Frederick Sanger ( 1918 -)Paul Berg: for his fundamental studies of the biochemistry of nucleic
42、 acids, with particular regard to recombinant-DNA. Walter Gibert( 1932- )Walter Gibert & Frederick Sanger: for their contributions concerning the determination of base sequences in nucleic acids. Introduction of DNA sequencing by the Sanger method based on the principle of DNA synthesis by DNA polym
43、erases; requires a primer, a template and DNA polymerase; 3-OH of the primer reacts with an incoming deoxynucleoside triphosphate (dNTP) to form a new phosphodiester bond. ddNTP terminates the DNA synthesis.The Sanger method is also calledthe dideoxy methodPrinciple of automating DNA sequencingddNTP
44、s are labeled with differently colored fluorescent tagsNext-generation DNA sequencing Illumina (Solexa) sequencing Nature Biotechnology 2008, doi: 10.1038/nbt1486 In vitro construction of a sequencing library; In vitro clonal amplification to generate sequencing fragments, DNA polymerase, primers, f
45、luorescently labeled nucleotides; used for analysis of genome and transcriptome; Higher efficiency and lower costs;Chemical synthesis of DNA by the phosphoramidite methodChemical synthesis of DNA by the phosphoramidite methodlength: 70-80 ntOther functions of nucleotidesGeneral structure of the nucl
46、eoside 5-mono-, di-, and triphosphatesAdenosine 5-triphosphate (ATP) carry chemical energy in cells ester bond (hydrolysis: 14kJ/mol) anhydride bond (hydrolysis: 30 kJ/mol)Coenzyme A (CoA) functions in acyl group transfer reactionsR-C-S-CoA=OAcyl-CoA The acyl group (such as the acetyl or acetoacetyl group) is attached to the CoA through a thioester linkage.NAD+ functions in hydride transfers, and FAD, in electron transfersSecond messengers play regulatory roles in cells(ATP is the substrate)(GTP is the substrate)(present in bacteria)
生物化學(xué)：Chapter 8 Nucleotides and nucleic acids
