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Download Chapter02 Nucleotides and Nucleic Acids(核酸化学)
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教 案 ~ 2007 学年 第一 学期 2006 学 院 教 名 称 研 生命科学学院 室 课 程 名 称 授 课 对 象 授 课 教 师 陈文利 称 副教授 职 教 材 名 称 2006 年 9 月 生物化学 2005 级生物技术专业 现代生物化学 日 授课题目(教学章、节或主题) : 教学器材 与工具 第二章 核酸化学 Nucleotides and Nucleic Acids 授课时间 多媒体设施、黑板与 笔 第 3,4,5 周一第 9-14 节 教学目的、要求(例如识记、理解、简单应用、综合应用等层次) : 核酸的组成成分及性质特点,用动画描述核酸的二级结构及主要的性质,要求学 生掌握核酸的二级结构特征及其稳定作用力。 教学内容(包括基本内容、重点、难点) : Outline Nitrogenous Bases The Pentoses of Nucleotides and NA Nucleosides are Formed by Joining a Nitrogenous Base to a Sugar Nucleotides - Nucleoside Phosphates Nucleic Acids are Polynucleotides Classes of Nucleic Acids Primary Structure of Nucleic Acids ABZs of DNA Secondary Structure Denaturation and Renaturation of DNA Tertiary Structure of DNA Chromosome Structure Chemical Synthesis of Nucleic Acids Secondary and Tertiary Structure of RNA Nitrogenous Bases Know the basic structures Pyrimidines – Cytosine (DNA, RNA) – Uracil (RNA) – Thymine (DNA) Purines – Adenine (DNA, RNA) – Guanine (DNA, RNA) Properties of Pyrimidines and Purines Keto-enol tautomerism Acid/base dissociations Strong absorbance of UV light Pentoses of Nucleotides Know these structures too D-ribose (in RNA) 2-deoxy-D-ribose (in DNA) The difference - 2'-OH vs 2'-H This difference affects secondary structure and stability Nucleosides Linkage of a base to a sugar Base is linked via a glycosidic bond The carbon of the glycosidic bond is anomeric Named by adding -idine to the root name of a pyrimidine or -osine to the root name of a purine Conformation can be syn or anti Sugars make nucleosides more water-soluble than free bases Nucleotides Nucleoside phosphates Know the nomenclature "Nucleotide phosphate" is redundant! Most nucleotides are ribonucleotides Nucleotides are polyprotic acids Functions of Nucleotides Nucleoside 5'-triphosphates are carriers of energy Bases serve as recognition units Cyclic nucleotides are signal molecules and regulators of cellular metabolism and reproduction ATP is central to energy metabolism GTP drives protein synthesis CTP drives lipid synthesis UTP drives carbohydrate metabolism Nucleic Acids - Polynucleotides Polymers linked 3' to 5' by phosphodiester bridges Ribonucleic acid and deoxyribonucleic acid Know the shorthand notations Sequence is always read 5' to 3' In terms of genetic information, this corresponds to "N to C" in proteins Classes of Nucleic Acids DNA - one type, one purpose RNA - various types, various purposes – ribosomal RNA (rRNA) - the basis of structure and function of ribosomes – messenger RNA (mRNA)- carries the message – transfer RNA (tRNA) - carries the amino acids – Other RNAs Small nuclear RNA (splicesomal RNA) Small nucleolar RNA (ribosomal RNA processing) Interference RNA (gene silencing) microRNA (translation regulation) Virus RNA (code virus genome) The DNA Double Helix Stabilized by hydrogen bonds! "Base pairs" arise from hydrogen bonds Erwin Chargaff had the pairing data, but didn't understand its implications Rosalind Franklin's X-ray fiber diffraction data was crucial Francis Crick knew it was a helix James Watson figured out the H-bonds The Structure of DNA An antiparallel double helix Diameter of 2 nm Length of 1.6 million nm (E. coli) Compact and folded (E. coli cell is only 2000 nm long) Eukaryotic DNA wrapped around histone proteins to form nucleosomes Base pairs: A-T, G-C Messenger RNA Transcription product of DNA In prokaryotes, a single mRNA contains the information for synthesis of many proteins In eukaryotes, a single mRNA codes for just one protein, but structure is composed of introns and exons Eukaryotic mRNA DNA is transcribed to produce heterogeneous nuclear RNA – mixed introns and exons with poly A – intron - intervening sequence – exon - coding sequence – poly A tail - stability? Splicing produces final mRNA without introns Ribosomal RNA Ribosomes are about 2/3 RNA, 1/3 protein rRNA serves as a scaffold for ribosomal proteins 23S rRNA in E. coli is the peptidyl transferase! Transfer RNA Small polynucleotide chains - 73 to 94 residues each Several bases usually methylated Each a.a. has at least one unique tRNA which carries the a.a. to the ribosome 3'-terminal sequence is always CCA Aminoacyl tRNA molecules are the substrates of protein synthesis DNA & RNA Differences? Why does DNA contain thymine? Cytosine spontaneously deaminates to form uracil Repair enzymes recognize these "mutations" and replace these Us with Cs But how would the repair enzymes distinguish natural U from mutant U? Nature solves this dilemma by using thymine (5-methyl-U) in place of uracil DNA & RNA Differences? Why is DNA 2'-deoxy and RNA is not? Vicinal -OH groups (2' and 3') in RNA make it more susceptible to hydrolysis DNA, lacking 2'-OH is more stable This makes sense - the genetic material must be more stable RNA is designed to be used and then broken down Hydrolysis of Nucleic Acids RNA is resistant to dilute acid DNA is depurinated by dilute acid DNA is not susceptible to base RNA is hydrolyzed by dilute base See the below Figure for mechanism Restriction Enzymes Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes" Type II and III restriction enzymes cleave DNA chains at selected sites Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage An enzyme that recognizes a 6-base sequence is a "six-cutter" Type II Restriction Enzymes No ATP requirement Recognition sites in dsDNA usually have a 2-fold axis of symmetry Cleavage can leave staggered or "sticky" ends or can produce "blunt” ends Type II Restriction Enzymes Names use 3-letter italicized code: 1st letter - genus; 2nd,3rd - species Following letter denotes strain EcoRI is the first restriction enzyme found in the R strain of E. coli Primary Structure Sequencing Nucleic Acids Chain termination method (dideoxy method), developed by F. Sanger Base-specific chemical cleavage, developed by Maxam and Gilbert Both use autoradiography - X-ray film develops in response to presence of radioactive isotopes in nucleic acid molecules DNA Replication DNA is a double-helical molecule Each strand of the helix must be copied in complementary fashion by DNA polymerase Each strand is a template for copying DNA polymerase requires template and primer Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA DNA polymerases add nucleotides in 5'-3' direction Chain Termination Method Based on DNA polymerase reaction Run four separate reactions Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P-32-labelled Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP Chain Termination Method Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide Chain Termination Method Run each reaction mixture on electrophoresis gel Short fragments go to bottom, long fragments on top Read the "sequence" from bottom of gel to top Convert this "sequence" to the complementary sequence Now read from the other end and you have the sequence you wanted - read 5' to 3' The ABZs of DNA Secondary Structure See the below Figure for details of DNA secondary structure Sugar-phosphate backbone outside Bases (hydrogen-bonded) inside Right-twist closes the gaps between base pairs to 3.4 A (0.34 nm) in B-DNA The “canonical” base pairs The canonical A:T and G:C base pairs have nearly identical overall dimensions A and T share two H-bonds G and C share three H-bonds G:C-rich regions of DNA are more stable Polar atoms in the sugar-phosphate backbone also form H-bonds Forces That Stabilize Nucleic Acid Double Helix There are two major forces that contribute to stability of helix formation – Hydrogen bonding in base-pairing – Hydrophobic interactions in base stacking Types of DNA Double Helix Type A: major conformation of RNA, minor conformation of DNA; Type B: major conformation of DNA; Type Z: minor conformation of DNA Major and minor grooves The "tops" of the bases (as we draw them) line the "floor" of the major groove The major groove is large enough to accommodate an alpha helix from a protein Regulatory proteins (transcription factors) can recognize the pattern of bases and H-bonding possibilities in the major groove Denaturation AND Renaturation of DNA When duplex DNA molecules are subjected to conditions of pH, temperature or ionic strength that disrupt hydrogen bonds, the strands are no longer held together. The double helix is denatured. If the temperature is the denaturing agent, the double helix is said to melt; The phenomenon that the relative absorbance of the DNA solution at 260 nm increases as the bases unstack is called hyperchromic shift; If one fellows the absorbance as a function of temperature, the midpoint temperature of the absorbance curve is termed melting temperature, Tm. Tm =81.5 oC+16.6 (log10[Na+])+0.41(fraction G+C)-0.63(% formamide)-(600/length) DNA of different sequences have different Tm. Tm is higher for DNA that contain more GC pairs; Tm is also directly proportional to the ionic strength of the solution. (salts can shield repulsions of negatively charged phosphate groups) Renaturation (or Annealing) Renaturation refers to the process of DNA strands associate into a double helix; Renaturation can be analyzed quantitatively: C is the amount of single stranded DNA remaining, C0 is the initial single stranded DNA. Structural Changes in DNA Melting nRenaturation refers to the process of DNA strands associate into a double helix; nRenaturation can be analyzed quantitatively: C is the amount of single stranded DNA remaining, C0 is the initial single stranded DNA. Ribosomal RNA Ribosomes synthesize proteins All ribosomes contain large and small subunits rRNA molecules make up about 2/3 of ribosome High intrastrand sequence complementarity leads to (assumed) extensive base-pairing Ribosomal RNA Secondary structure features seem to be conserved, whereas sequence is not There must be common designs and functions that must be conserved 重点:核酸的组成成分及性质特点用动画描述核酸的二级结构及主要的性质,要求学 生掌握核酸的二级结构特征及其稳定作用力 难点:DNA 二级结构特点,DNA 一级结构测定 双脱氧法(酶法)--原理 教学过程设计(要求阐明对教学基本内容的展开及教学方法与手段的应用、讨论、作业布置): 利用课件结合板书介绍核酸化学的基础知识,了解核酸化学的研究新进展,与蛋白质相比较 重点掌握核酸二级结构特征及等电点等主要性质,在理解的基础上布置作业,让学生在作业中发 现问题提出问题,对于比较难理解的老师在课堂上再次强调。在教学过程中给学生介绍学习方法 及鼓励学生拓展知识。