分子生物学

1.1.1 DNA+ deoxyribonucleic acid;+ function: The genetic information carrier,

responsible for the genetic information storage and transmission ；

+ distribution ：+ Eukaryotes: nucleus （ 98% ） ,organelle （ 2% ）；+ prokaryotes: nucleoid 、 plasmids+ viruses

1.1 type and distribution of nucleic acid

• ribonucleic acid ；• Function: mainly participate the expression of

genetic information• Type: rRNA （ ribosomal RNA ） mRNA （ messenger RNA ） tRNA （ transfer RNA ）• distribution: cytoplasm(90%), nucleus(10%) ；

viruses

mRNA

rRNA

tRNA

• 种类最多，比例最低，寿命最短；• Each polypeptide has corresponding

mRNA ， the difference among different mRNA

is quite big(in length and molecular weight) ；• Functions: as intermediaries, carrying genetic

information from genes to ribosomes, where the

corresponding proteins can be synthesized (as

templates).

• 分子量最小；• Functions: as adapter molecules (transport

amino acids), that faithfully translate the information in mRNA into a specific sequence of amino acids ；

• 每个 Aa 至少有一个对应的 tRNA （如丙氨酸tRNA 、 tRNAAla ）。

• 不同 tRNA 分子的大小很相似， 73-78Nts

• 比例最大，分子量最大；• Functions: structural components of

ribosomes, the complexes that carry

out the synthesis of proteins

名称 缩写 功能

核糖体 RNA rRNA 核糖体组成成分

信使 RNA mRNA 蛋白质合成模板

转运 RNA tRNA 转运活化氨基酸

不均一核 RNA hnRNA 成熟 mRNA 的前体

核内小 RNA snRNA 参与 hnRNA 的剪接转运

核仁小 RNA snoRNA 参与 rRNA 的加工和修饰

胞质小 RNA scRNA蛋白质内质网定位合成信号的识别组分

核酸是生命遗传信息的携带者和传递

者（生命的延续、遗传特性的保持、

生长发育、细胞分化）

The RNA world ， the DNA core

nucleic acid

nucleotide

phosphoric acid nucleoside

pentose base

means pentose, DNA has deoxyribose, RNA has ribose ；

means nitrogenous bases

means phosphate

nucleotide

ribose

（ in RNA ）

2’- deoxyribose

（ in DNA ）

purine ring

N

N65

432

1

9

N

N N

N12

34

65

8

7

Pyrimidine ring

DNA

RNA

• The chemical modification of bases is

widespread, and has a number of specific

roles.

• Such as: the methylation of the N-6 position

of A 、 the 4-amino group and the 5-

position of C

2.3.1 nucleoside ：• DNA: 2’-deoxyribose + A 、 T 、 C 、 G

• RNA: ribose + A 、 U 、 C 、 G

• The base is linked to position 1 on a pentose

by a glycosidic bond from N1 of pyrimidine

or N9 of purine

N

NN

N

NH

H

NH

NH

O

O

O

OHOH

OH

OHCH2

H2O

P OH

O

O

O

H2O

base

phosphoric acid

pentose

glycosidic bond

Phosphate bond

1.2.3.2 nucleotide

2.1 primary structure of nucleic acid• definition ： the nucletide residue sequence of

the polynucleotide chain ；• linkage ： 3’,5’-phosphodiester bond ；• backbone ： phosphate + pentose ；• direction ： from the 5’ terminus to the 3’

terminus ；• representation

A

P

C

P

T

P

C

P

G

P

A

PP

OH5'3'

5'pApCpTpCpGpA-OH3'

5'ACTCGA3'

• Three discoveries (physical chemistry studies

and acid and alkali titrate studies on DNA base 、

chargaff rules 、 x-ray photograph of

DNA)converged in the construction of the

double helix model for DNA by Watson and Crick

in 1953

• means DNA double helix

• the base composition of DNA varies from one species to another, while DNA specimens isolated from different tissues of the same species have the same base composition ；

• in nature DNA,the number of G units equals the number of C units and the number of A units equals the number of T units ( this strongly hinted towards the base pair makeup of DNA ) ；

• The base composition of DNA in a given species does not change with an organism’s age,nutritional state,or changing environment 。

Chargaff rules ：

• X~ray photograph of DNA with high quality ：

DNA specimens from different species have the

same results （ constant width;3.4nm ）；• Chargaff rules ： the rule of the composition of

DNA

• physical chemistry studies and acid and alkali

titrate studies on DNA base ；

• Two polynucleotide chains in a DNA double helix

• antiparallel （ one strand runs in the 5’→3’ direction,whlie its partner runs 3’→5’ ）

• Along the same axis ， two chains are wound around each other, resulting in a right-handed double helix

• forms a major groove and a minor groove

2.2.1 key notes of DNA double helix

• The bases lie on the inside ， the sugar-phosphate backbone is on the outside

• The bases are flat structure, lying in pairs perpendicular to the axis

• The diameter of the

double helix is 2nm

• There is a complete

turn every 3.4nm,

with 10bp per turn.

• Two chains in the double helix associate by hydrogen bonding between the bases,G with C and A with T(base pairing , complementary).

• Three hydrogen bond can form specifically between G and C,but only two can form between A and T;

1. Two

chains,antiparallel, right-

handed, major groove

and minor groove2. outside(sugar-phosphate backbone), inside(bases), 3’,5’-phosphodiester bond

3. 2nm in diameter, 10bp per turn, 3.4nm

4. Base pairing

小沟

大沟

5’

3’ 5’

3’

小沟

• Explains for the first time how genetic information is encoded in DNA and passed from one generation to the next ；

• tremendous strides in our understanding of the structure of DNA and, as a result our ability to work with and manipulate the information-rich DNA molecule

• base pairing → DNA semi-conservative replication mechanism

• In physiological conditions,the DNA double helix is very stable.

stable factors ：• H bonding between complementary base pairs ；• Base-stacking interaction （ hydrophobic effect, the

major factor ）；• electrovalent bond （ between the negative charges

carried on the phosphate groups and the positive charges carried on the proteins or metal ions ）

Base Obliquity

helix rise per base pair

bp numberper turn

Helical sense

diameter（ nm ）

B-form

Z-form

0-1

19-20

9

0.34 nm  

0.23 nm 

0.38 nm

10 

11 

12

R handed 

R handed 

L handed

2.0-2.37 

2.55 

1.8-1.84

A-form

B-form ： relative humidity is 92%

A-form ： relative devoid of water (under 75%)

Z-form ： left handed helix

H-form ： triple helix

H-forms

• Pentose is ribose ， T is replaced by U ；• 大多数 RNA 分子是单链，不遵守碱基的数量

比例关系• 局部的碱基互补配对 + 不能配对的部分—

hairpin or internal loop or bulge ；• RNA 分子是含短的不完全的螺旋区的多核苷

酸链。

Prokaryote mRNA ：

polycistron

/

/

Eukaryote mRNA ：

monocistron

cap in 5’ terminus

polyA in 3’ terminus

cistron ： mRNA 上能够翻译出一条多肽链的核苷酸功能片段。

polyA ：指 20~250 个多聚腺苷酸。

cap ：甲基化（ 碱基）的鸟苷酸，通过焦磷酸与另一个发生了甲基化（核糖）的核苷酸以 5’,

5’- 三磷酸相连。

/

//

/

/

�

�

5

4

32

1H

OH

CH2 O P

O

O

O

P

O

O P

O

O-OO�

1

9

8

765

42

N+

N

NH

N

CH3

O

H2N

O

OH

H

N

H

O

CH2

H H1

23

4

5

�

/

/

/ /

OH

O

--

P O-O

O-AAAAAAA_OH

“帽子”结构：

G 甲基化

焦磷酸OCH3

甲基化

H

任意碱基

1

2

3

Four stems

Modifiedbases

cloverleaf

4

Four loops

Aa acceptor arm

• 包含有 3’- 端和 5’- 端• 3’- 端是 CCA

• 功能：携带活化 Aa

Anticodon arm

• 环正中的 3 个核苷酸残基称为 anticodon

• 功能：通过反密码子与mRNA 上密码子的配对，来决定 Aa 的正确位置

D arm• 环： 8-12 个核苷酸• 臂： 3-4bp• 功能：氨酰 -tRNA 合

成酶结合位点TC arm • 此环中基本含有 TC• 功能：结合核糖体Variable loop• 变化较大，是不同

tRNA 的区别之处

• 在二级结构的基础上，突环上未配对的碱基通过整个分子的扭曲而配成对

• 已知 tRNA 的三级结构均为倒 L 形。

3.1 chemical and physical properties of nucleic acid

• 颜色形状： DNA 为白色纤维， RNA 为白色粉末；• 溶解度：微溶于水，钠盐的溶解度较高。不溶于

有机溶剂（用乙醇从溶液中沉淀核酸）；

DNA 核蛋白 RNA 核蛋白

0.14mol/L NaCl - +

1-2mol/L NaCl + -

• viscosity ： DNA solutions have a high

viscosity, while RNA decreases sharply

• 颜色反应：

• DNA （ 2-deoxyribose ） + 二苯胺在酸性条件下形成兰紫色化合物；

• RNA （ ribose ） + 苔黑酚在浓盐酸和加热条件下形成绿色化合物。

• according to substrates ： DNases 、 RNases• according to function methods ：

exonucleases 、 endonucleases• exonucleases ： catalyze the hydrolysis of single

nucleotides from the end of a DNA or RNA chain ；• endonucleases ： catalyze the hydrolysis of bonds

between nucleic acids in the interior of DNA or RNA sequence. Restricition endonuclease recognizes specific DNA sequences and cut the DNA backbone at a specific site within that sequence

Palindromis sequence

Restriction enzymes may give products with 5’- or 3’- tails or blunt ends

Sticky ends

• The conjugated aromatic

of bases→nucleic acid

absorb UV

• λmax=260nm

• The UV absorption

properties of nucleic acids

can be used for

detection,quantitation

and assessment of purity

• RNA ： OD260 / OD280=2.0

• DNA ： OD260 / OD280=1.8

The value greater than 1.8,suggests RNA contamination

less protein

• OD260 = 1.0 = 50μg/mL dsDNA

= 40μg/mL ssDNA/RNA

= 20μg/mL (d)NTP

3.4.1 denaturation

3.4.1.1 definition: a number of

physical and chemical factors

can lead to the destruction of

double-stranded hydrogen-

bonded regions of DNA and

RNA,the double-stranded

nucleic acids are converted to

single strands.

3.4.1.2 characters of denatural DNA ：• biological activity changed(even lost) ；• The UV absorption value increased

(hyperchromicity),viscosity decreased ， solubility decreased,buoyant density increased ；

• The structure became loose,easy to be hydrolyzed ；• Molecular weight remained unchanged

• Increased temperature can bring about

DNA denaturation;

• 将 DNA 的变性达到 50% 时的温度称为

DNA 的解链温度（ melting temperature,

Tm ） , 也称熔解温度或 DNA 的熔点。

• Tm 是 DNA 的特征常数。

• G+C content ： G 和 C 的含量高， Tm 值高。通过测定 Tm 值，可反映 DNA 分子中 GC 含量：

（ G+C ） %= （ Tm-69.3 ） ×2.44

• DNA 的均一性：均一性较好，则 Tm 值范围较小；

• 介质的离子强度： DNA 应保存在含盐的缓冲液中

3.4.2.1 definition ：热变性 DNA 在温度逐渐降低时，在一定浓度的盐溶液中，两条分开的单链可以重新恢复双螺旋结构，又称为退火（ annealing ） 。

3.4.2.2 characters of renaturation• biological activity resumed• hypochromicity• viscosity increased, solubility increased,

buoyant density decreased

3.4.2.3 factors that influence renaturation

• temperature ：缓慢比快速好

• DNA concentration ：越高越快

• Size of DNA segments ：片段越大，越不容易

• DNA complexity ：重复片段 (repetitive segment)

越多复性速度越快，单重复片段复性速度最慢

• 定义： the renaturation

of regions of

complementarity

between different

nucleic acid

strands(DNA or RNA)

• 特点：灵敏度高、专一性强

1