Download Chapter02 Nucleotides and Nucleic Acids(核酸化学)

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。