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Chapter two Structures and Functions of Nucleic Acids 目录 Main contents Introduction 1. The components of nucleic acids 2. The structures and functions of DNA 3. The structures, and functions of RNA 4. The physicochemical properties of nucleic acids 5. Nucleases and Ribozymes 6. Genomics and Human Genome Project Disease cases Demands Questions 目录 Nucleic acid Polynucleotide or polymers of nucleoties which can carry, transmit and express the genetic information 目录 1.The discovery of nucleic acids and the research advance • 1868 Fridrich Miescher the isolation of nuclein from pus leukocytes • 1944 Avery the confirmation of DNA as genetic molecules • 1953 Watson and Crick the discovery of DNA double helix structure • 1968 Nirenberg the discovery of genetic codons • 1975 Temin and Baltimore reverse transcriptase • 1981 Gilbert and Sanger the method for DNA sequencing (dideoxy chain terminator sequencing) • 1985 Mullis the creation of PCR technology • 1990 human genome project (HGP) start-up in America • 1994 Chinese human genome project start-up • 2001 HGP were nearly finished 目录 2. The sorts and distribution of nucleic acids Deoxyribonucleic acid, DNA more than 90% in nucleus , and other found outside of nucleus such as mitochondria, chloroplast, plasmid Carry genetic information, determine the genotype of individual or cell Ribonucleic acid, RNA Distributed mainly in cytoplasm, minority in nucleus Transmit the genetic information and gene expression , sometime work as the carrier of genetic information such as RNA virus 目录 Section One The Monomeric Units of Nucleic Acids Nucleotides 目录 Chemical composition of nucleic acids 1. Element component C、H、O、N、P(9~10%) 2. Molecular component —— base:purines,pyrimidines —— ribose:ribose,deoxyribose —— phosphate 3. Building units (monomeric units): nucleotides 目录 Chemical composition of nucleic acid The composition of nucleotide nucleic acid base ribonucleotide Pentose ribonucleoside phosphate phosphate base Pentose Elementary unit of nucleic acid is nucleotide DNA: dAMP, dGMP, dCMP, dTMP RNA: AMP, GMP, CMP, UMP 目录 NH2 Bases N N Purine N 7 8 9 NH NH 5 4 6 3 N 1N 2 N adenine, A O N NH guanine, G NH N NH2 目录 O Pyrimidine 5 4 NH N 3 2 6 1 NH NH O uracil, U O NH2 H3 C NH N NH cytosine, C O NH O thymine, T 目录 Pentose HO CH 2 5´ O OH HO CH 2 OH O 1´ 4´ 3´ 2´ OH ribose (RNA) OH OH deoxyribose (DNA) 目录 1. The structures of nucleotides (1) The formation of ribonucleoside It can be formed by glycoside bond joined base to ribose (ribonucleoside) or deoxyribose HO CH2 (deoxyribonucleoside) NH2 N 1 O N O 1´ ribonucleoside :AR, GR, UR, CR OH OH deoxyribonucleoside :dAR, dGR, dTR, dCR 目录 目录 (2) The naming of nucleoside or nucleotides 目录 目录 目录 Some important free nucleotide and their ramifications in organisms nucleotides:NMP,NDP,NTP cyclic nucleotide: cAMP,cGMP Biological active substances containing nucleotide: NH2 NAD+、NADP+、CoA-SH、FAD etc, containing N AMP NH NH NH22 2 N NN O N NN O OO OO NN N CH HO PHOO PPHO OO CH CH 22O OO PPP O NN 2O N O OH OH OH OH OH OH cAMP ADP AMP ATP NAD+ O OH OH OH OH OH OH O CH2 P O N O OHNADP+ OH N N 5´end C (3)The linkage of nucleotides The nucleotides A are linked together by phosphodiester bonds to form polynucleotides, namely nucleic acids G 3´end 目录 5′end 2. Primary Structure of nucleic acids C Definition The linear sequence of (deoxy)nucleotides ( or base sequences ) in DNA (RNA) is termed primary structure of DNA (RNA). Linkage bond phosphodiester linkage A G 3′end 目录 writing A 5 P G P T P G P C P T P OH 3 5 pApCpTpGpCpT-OH 3 5 A C T G C T 3 目录 1.2 Properties of nucleosides and nucleotides Tautomerism between the lactam and lactim Absorbance of ultraviolet light at 260 nm 目录 Section Two Dimensional Structure and Function of DNA 目录 目录 • The secondary structure of DNA----double helix structure – The research background and historic significance of DNA helix structure – The key points of DNA double helix structure • The superhelix structure of DNA and the its composition in chromatin – The DNA superhelix structure – The spatial structure of DNA in prokaryote – DNA constitution in the nucleus of eukaryote cells • DNA functions 目录 5′end 1. Primary Structure of nucleic acids C Definition The linear sequence of deoxynucleotides ( or base sequences ) in DNA is termed primary structure of DNA. Linkage bond phosphodiester linkage Backbone: ---Pi-dR-Pi-dR--- A G 3′end 目录 2. Secondary Structure of DNA---Double Helix Model 目录 2.1 Background The analysis of base composition of DNA Chargaff rules The analysis of chemical and physical data on DNA building model Primarily x-ray diffraction data collected by Rosalind franklin and Maurice Wilkins 目录 Chargaff rules a. A = T, G = C; b. The base composition is different in different organism species. c. The base composition of different organs in same individual is same. 目录 2.2 The key points of DNA Double Helix Model (1) The DNA double helix is oriented to right-handed running. (2) The two DNA strands minor groove are organized in an antiparallel arrangement (i. e. The two strands run in major groove opposite directions, one strand is oriented 5’3’ and the other is oriented 3’ 5’ ). 目录 3’ 5’ 5’ 3’ 目录 2.2 The key points of DNA Double Helix Model (3) The bases of the two strands form hydrogen bonds to each other; A pairs with T and G pairs with C. For each round of the helix, there are 10 pairs of bases 目录 3’ 5’ minor groove major groove 5’ 3’ Base pairing rules A T Hydrogen bond A=T G≡C C G Hydrogen bond 目录 2.2 The key points of DNA Double Helix Model (4) The stable forces are hydrogen bonds between base pairs and base stack force. (5) There are major grooves and minor grooves in DNA double helix. 3’ 5’ minor groove major groove 5’ 目录 3’ The summary on DNA double helix (Watson, Crick, 1953) (1) right-handed oriented double helix, antiparallel (2) Backbone outside, bases inside (3) base-pairing, A=T, G≡C (4) A running of the helix containing 10 pairs of bases hydrogen bonds, base force (5) Structure stable depends on stack the vice-bonds (6) There are minor grooves and major grooves 目录 . Polymorphism of secondary structure of DNA 2 4 目录 Forms of DNA Form Pitch(nm) Residues per turn inclination of base pair from horizontal A 2.8 11 20° B 3.4 10 0° Z 4.5 12 7° 11 20° RNA-DNA hybrid 2.8 Source: From Davidson, The biochemistry of the Nucleic Acids, 8thed., revised by Adams, et al. Copyright ©Chapman & Hall, London. 目录 2.5 The Superhelix Structure of DNA (1)Superhelix structure of DNA in prokaryote ------Circular double stranded superhelical DNA 目录 (2)Superhelix structure of DNA in eukaryote ------Nucleosomes The nucleosome in eukarytic cells DNA double helix superhelix nucleosome chromatin chromosome The nucleosomes in eukarytic cells consist of DNA and proteins 目录 The elementary unit of chromosome is nucleosome which consists of DNA and histone. Histone includes H1, H2A, H2B, H3, H4. Two molecules of each H2A, H2B, H3, H4 constitute the core particle of nucleosome. 目录 Core particles Linkage DNA H1, H2A, H2B, H3, H4 histones 目录 目录 10 nm fiber lengthwise section Transv erse section 30 nm fiber 300nm solenoid chromosome chromatin 目录 The folding of nucleosomes, the chromatin condensation, the formation of chromosome 2.6 Functions of DNA Genes consist of regulatory region and large protein-coding segments. Genome is a whole sequence of DNA in an organism. Genetic code: The letters A,G,T and C correspond to the nucleotides found in DNA. They are organized into three-letter code words called codons, and the collection of these makes up the genetic codes. 目录 DNA functions: 1. Template of replication 2. Template of transcription 3. To accept some mutations 目录 Section Three Structures and Functions of RNA 目录 Structure and Function of RNA 目录 1. Structure of RNA The primary structures of RNA belong to single stranded linear polynucleotide, but contain part self-complementary pairing, namely hairpin structures Elementary unit of nucleic acid is nucleotide RNA: AMP, GMP, CMP, UMP 目录 2. Types and Functions of RNAs 2.1 Messenger RNA Structure Characters of mRNA: (1) 5’-cap sequence: m’GpppNm The cap functions are to boost the binding of ribosome with mRNA, and to increase the stability of mRNA. 目录 Cap structure of mRNA 5’-end Guanine 目录 Structure Characters of mRNA: (2) 3’-end sequence: poly A The 3’-end functions are related to increase the stability of mRNA and the lifetime worked as template for translation. 3’ poly A 5’ cap structure 5’ uncoding region coding region 3’ uncoding region 目录 (3) Mature process of mRNA * (intron) (exon) hnRNA mRNA 目录 (4) mRNA functions: As a template of protein synthesis, it contains triplet codes. So, mRNA carries the information for the primary structure of proteins, serves as template of protein synthesis during translation. 目录 intron Eukaryotic 真核细胞cells Prokaryotic cells 细胞质 extron 外显子 细胞核 内含子 DNA 转录 Transcript DNA Transcript 转录 hnRNA 转录后剪接 Prost-transcript 转运 modification mRNA 翻译 translation 蛋白 protein mRNA protein 蛋白 翻译 translation 目录 2.2 Transfer RNA (tRNA) (1) Composition of transfer RNA, tRNA It’s the smallest RNA among RNAs, only consists of 70~90 nucleotides. tRNAs contain some unusual (modified) bases, such as 7-methylguanine, pseudouridine, dehydrouridine, 目录 O N NH H H NH N N CH3 H H CH3 N,Ndimethylguanine HN N NH O unusual bases CH2 CH C CH3 CH3 NH NH O Dihydrouridine S NH N N N6-isopreneadenine NH O 4-thiouracil 目录 (2) Functions of transfer RNA, tRNA Two roles: Activating amino acids Recognizing codons in mRNA The tRNA molecules serve as adapters for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids. 目录 (3) The structure characters of tRNA ◆Contain rare bases, such as DHU, pseudouridine ( ), mG, mC ◆ Stem-loop structure ( local double strands) Secondary structure: cloverleaf pattern ◆ Anticodon in the anticodon loop ◆ The base sequence of an anticodon can reversely complement with codon on mRNA. 目录 Amino acid arm TΨC loop DHU loop variable loop The cloverleaf pattern of tRNA ( secondary structure) Anticodon loop 目录 The anticodon at the end of a basepaired stem recognizes the triplet nucleotide or codon of the template mRNA. 5’ AUG UAC GCA CCA UCG Met AUG mRNA 3’ 5’ 5’ Ala 5’ 3’ Tyr Pro 目录 The cloverleaf pattern of tRNA ( secondary structure) 目录 The tertiary structure of tRNA 目录 2.3 Ribosomal RNA (rRNA) (1) rRNA structure (2) rRNA functions a. A component of ribosomes b. Ribosomes work as the apparatus of protein synthesis 目录 (3) rRNA sorts ( dependent on the sedimentation coefficient, S) Eukaryotes Prokaryotes 5S rRNA 28S rRNA 5S rRNA Large subunit 5.8S rRNA 18S rRNA 23S rRNA 16S rRNA Large subunit small subunit small subunit 目录 The components of ribosome Prokaryote ( E coli.) Eukaryote (mouse liver) 30S 40S Small subunit rRNA 16S 1542 nucleotides 18S 1874 nucleotides proteins 21 Occupy 40% of total weight 33 Occupy 50% of total weight Large subunit 50S 60S rRNA 23S 5S 2940 nucleotides 120 nucleotides 28S 5.85S 5S 4718nucleotides 160nucleotides 120nucleotides proteins 31 Occupy 30% of total weight 49 Occupy 35% of total weight 目录 2.4 Other Small Stable RNA A large number of discrete, highly conserved, and small stable RNA species are found in eukaryotic cells. The majority of these molecules exist as ribonucleoproteins and are distributed in the nucleus, in the cytoplasm, or in both. 目录 The major sorts of RNA and their functions In the nucleus or in the cytoplasm Ribosomal RNA Messenger RNA Transfer RNA rRNA mRNA tRNA mitochondrion mt rRNA mt mRNA mt tRNA heterogeneous nuclear RNA hnRNA Small nuclear RNA snRNA Small nucleolus RNA SnoRNA Small cytoplast RNA scRNA/7SL-RNA functions the components of ribosomes the template for translation activating AA and recognizing codons on mRNA the precursor of mRNA related to the splicing and transfering of hnRNA related to the processing and modifying of rRNA the components of signal discriminator for proteins located in endoplasmic reticulum 目录 Small nuclear RNAs are significantly involved in mRNA processing and gene regulation. Of the several snRNAs, U1, U2, U44, U5, and U6 are involved in intron removal and the processing of hnRNA into mRNA. hnRNA ( heterogeneous nuclear RNA ) snRNA ( small nuclear RNA) 目录 Section Four The Properties of Nucleic Acid 目录 1. The general properties of nucleic acids 1.1 1.2 1.3 1.4 Acidic molecules Macromolecules High viscosity (DNA) Ultraviolet absorption ( 260 nm) 目录 Adenine Extinction coefficient Uracil Cytosine Guanine Thymine Wavelength Ultraviolet absorbent spectrum of various bases (pH 7.0) 目录 2. DNA denaturation Definition Double helix of DNA Single strand The denaturation factors heating, chemical reagents, ultraviolet light 目录 The essence of DNA denaturation is the breaking of hydrogen bonds between the double strands of DNA heating Cooling slowly Natural DNA Denatural DNA 目录 The changes of DNA properties after denaturation * Hydrogen linkage broken * A260 ( hyperchromic effect ) * Double helix single strand * Tm ( melting temperature ) * Tm and G + C Tm = 69.3 + 0.41 ( % G+ C ) < 20 bp, Tm = 4 ( G + C ) + 2 ( A + T ) 目录 The application of OD260 (1) The determination of the amount of DNA or RNA OD260=1.0 is corresponded to 50μg/ml double stranded DNA 40μg/ml single stranded DNA(or RNA) 20μg/ml oligonucleotides (2) To estimate the purification of nucleic acid samples Purified DNA : OD260/OD280 = 1.8 Purified RNA: OD260/OD280 = 2.0 目录 For example: the change of ultraviolet absorption spectrum of DNA induced by denaturation Absorbent value Denatured state Natural state Wave length The ultraviolet absorption spectrum of DNA Hyperchromic effect : the phenomenon of OD260 increase companied by DNA denaturation in solution 目录 Heated denaturation of DNA Melting curve: a graph in which absorbance versus temerature is plotted Temperature-optical density profile for DNA 目录 Tm: Tm is the temperature at which 50% of DNA in solution are denatured by heated , or called melting temperature, Tm. The value of Tm is related to the contents of G and C. 目录 3. The renaturation of DNA and nucleic acid hybridization (1) Renaturation Single strand of DNA double helix ( annealing ) The best annealing temperature: 25℃ < Tm 目录 Renaturation process denaturation Various source DNA renaturation Hybridization double stranded molecules of DNA-DNA 目录 (2) Nucleic Acid Hybridization Definition The process of forming a double stranded structure from two polynucleotide strands ( either DNA or RNA) from different source is termed hybridization Hybridization Principle ( To see the above slice or next slice) 目录 Double stranded DNA 1 Heterogeneous double stranded DNA single stranded DNA heating cooling Double stranded DNA 2 slowly Double stranded DNA single stranded DNA heating Adding marked single stranded DNA probes Forming the marked heterogeneous stranded DNA Cooling slowly The denaturation and renaturation of DNA, nucleic acid hybridization 目录 (3) The application of Hybridization technology ★ Determining whether a certain sequence occurs more than once in the DNA of a particular organism ★ Demonstrating a genetic or evolutionary relationship between different organisms ★ Determining the number of genes transcribed in a particular mRNA ★ Determining the location of any given DNA sequence by annealing with a complementary polynucleotide probe 目录 Section 5 Nucleases catalytic RNA and DNA 目录 1. Nucleases (1) Definition Nucleases are the enzymes capable of degrading nucleic acids. (2) Catalogue DNase Those which exhibit specificity for deoxyribonucleic acid are referred to as deoxyribonucleases RNase Those which specifically hydrolyze ribonucleic acids are ribonucleaes. Endonuclease Exonuclease 目录 Classification according to the substrates •deoxyribonuclease, DNase Specially degrade DNA •ribonuclease, RNase Specially degrade RNA。 –Classification according to the cut sites Endonuclease restriction endonuclease (RE) and nonRE Exonuclease 5’-3’ exonuclease or 3’-5’ exonuclease 目录 2. Ribozymes Ribozymes are RNA molecules with catalytic activity There are five classes of ribozymes ★ Three classes carry out self-processing reactions. ★ Two classes ( RNase P and rRNA )are true catalysts that act on separate substrates (containing proteins) 目录 3. Deoxyribozymes Some single-stranded DNA molecules are capable of adopting intricate tertiary structures and performing efficient catalysis. These special DNA sequences with catalytic capability are called deoxyribozymes. At present, deoxyribozymes are only found in laboratories. 目录 Section 6 Genomics and the Human Genome Project 目录 1. Characteristics of Eukaryotic Genome DNA Genomes in eukaryotes are much larger than in prokaryotes Only a few percent ( 2-4%) of DNA in a mammalian cell may suffice for all genes. Eukaryotes genes do not overlap but are spaced, on the average, 40 000 bp apart. There are split genes that consist of exons and introns in eukaryote genomes. 目录 2. The human Genome Project 1990, the project started An international program Make a sequence map for the total 3 billion bp of human genome Resource for the investigation of hereditary diseases as well as normal gene structure and expression 目录 Disease Cases 1. Hemoglobin diseases (血红蛋白病) 2. Horsebean disease (蚕豆病) 3. X-Fragile Syndrome (X-脆性综合征) 目录 Demands ♣Types of nucleic acids and their functions ♣ Basic units for nucleic acid composition ♣ The differences between DNA and RNA ♣ Secondary structure characters of DNA ♣ Structure characters of mRNA, tRNA, rRNA ♣ Physicochemical properties of DNA, RNA 目录 Questions: 1. Describe the sorts, functions of nucleic acids in nature. What are the characteristics of their chemical components or structures respectively? 2. What are the double helix of DNA? How to prove the structural characters of DNA double helix? 3. Why is it said that DNA are the carriers of genetic substances ? How to demonstrate it ? 目录 4. Which important physical and chemical properties of nucleic acids are there that can be utilized ? 5. Illustrate those important free mononucleotides existing in organisms 6. Do you know how to compare DNA with RNA? 7. What are the differences between nucleases and ribozymes? 目录 Friedrich Miescher (1844-1895) Friedrich Miescher worked at the Physiological Laboratory of the University of Basel and in Tübingen and is most well known for his discovery of the nucleic acids. 目录 Friedrich Miescher(1844-1895) From an early age Friedrich was recognized as being highly intelligent, but he was shy and introspective - perhaps in part as the result of a serious hearing impairment he had suffered from since boyhood. Despite this handicap he took great interest in music. Miescher had originally intented to study lymphocytes, he collected laudable pus from used bandages in the nearby hospital. He obtained a precipitate from the cell nuclei solution. Obviously this material must have come from the nucleus, and he therefore named it nuclein. Using elementary analysis, one of the few methods available to characterize an unknown compound. He extracted high-molecular-weight DNA from damaged cells eventually. 目录 Oswald T. Avery (1877-1955). Courtesy of The Rockefeller University Archives. Reichard, P. J. Biol. Chem. 2002;277:13355-13362 目录 O.T. Oswald Theodore Avery (1877~1955) Oswald Theodore Avery (1877-1955) was a distinguished bacteriologist and research physician and one of the founders of immunochemistry. He is best known, however, as a discoverer that deoxyribonucleic acid (DNA) serves as genetic material. The Oswald T. Avery Collection is a part of the Joshua Lederberg Papers, which are at the National Library of Medicine and available digitally. The collection was assembled by Nobel laureate Dr. Lederberg because of the strong connection between Dr. Avery's work and his own. The work of Avery and his lab, observes Dr. Lederberg, was "the historical platform of modern DNA research" and "betokened the molecular revolution in genetics and biomedical science generally. 目录 Rosalind Franklin - ( 19 20 19 58 ) 目录 Rosalind Franklin(1920-1958) Born in July of 1920, Rosalind Franklin graduated from Cambridge University and in 1951 went to work as a research associate for John Randall at King's College. A chemist by training, Franklin had made original and essential contributions to the understanding of the structure of graphite and other carbon compounds. It was Franklin alone whom Randall had given the task of elucidating DNA's structure. The technique with which Rosalind Franklin set out to do this is called Xray crystallography. With this technique, the locations of atoms in any crystal can be precisely mapped by looking at the image of the crystal under an X-ray beam. By the early 1950s, scientists were just learning how to use this technique to study biological molecules. Rosalind Franklin applied her chemist's expertise to the unwieldy DNA molecule. After complicated analysis, she discovered (and was the first to state) that the sugar-phosphate backbone of DNA lies on the outside of the molecule. She also elucidated the basic helical structure of the molecule. 目录 More advanced reading: • Nelson, D. L., and Cox, M. M. (2000) Lehninger Principles of Biochemistry, third edition, Worth Publishers. • Berg, J. M., Tymoczko, J. L., and Stryer, L. (2002) Biochemistry, Fifth edition, W. H. Freeman and Company, New York. • Mathews, C. K., van Holde, K. E., and Ahern, K. G. (2000) Biochemistry, third edition, Benjamin/Cummings (www.aw-bc.com/mathews/). 目录