目 录

Chapter two

Structures and Functions Structures and Functions of Nucleic Acidsof Nucleic Acids

目 录

Main contents

1. The components of nucleic acids

2. The structures and functions of DNA3. The structures, and functions of RNA4. The physicochemical properties of nucleic acids

5. Nucleases and Ribozymes

6. Genomics and Human Genome Project

Disease cases

Questions

Demands

Introduction

目 录

Nucleic acid

Polynucleotide or polymers of nucle

oties which can carry, transmit and exp

ress the genetic information

目 录

• 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

1.The discovery of nucleic acids and the research advance

目 录

2. The sorts and distribution of nucleic acids

more than 90% in nucleus , and other found outside of nucleus such as mitochondria, chloroplast, plasmid

Distributed mainly in cytoplasm, minority in nucleus

Deoxyribonucleic acid, DNA

Ribonucleic acid, RNA

Carry genetic information, determine the genotype of individual or cell

Transmit the genetic information and gene expression , sometime work as the carrier of genetic information such as RNA virus

目 录

The Monomeric Units of Nucleic Acids

Section One

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

nucleic acid ribonucleotide

ribonucleosidephosphate

base

Pentose

Elementary unit of nucleic acid is nucleotideDNA: dAMP, dGMP, dCMP, dTMPRNA: AMP, GMP, CMP, UMP

The composition of nucleotide

base

Pentose

phosphate

目 录

Purine

N

N

NH

N1

23

4

567

89

N

N

NH

N

NH2

adenine, A

N

NH

NH

N

NH2

O

guanine, G

Bases

目 录

N

NH1

32

45

6

Pyrimidine

cytosine, C

N

NH

NH2

O

uracil, U

NH

NH

O

O

thymine, T

NH

NH

O

O

CH3

目 录

Pentose

（ RNA ）

1´

2´3´4´

5´OH

O

CH2OH

OH OH

ribose（ DNA ）

OHO

CH2OH

OH

deoxyribose

目 录 deoxyribonucleoside ： dAR, dGR, dTR, dC

R

1. The structures of nucleotides

(1) The formation of ribonucleoside

It can be formed by glycoside bon

d joined base to ribose (ribonucleosid

e) or deoxyribose (deoxyribonucleosid

e) OHO

CH2

OHOH

N

N

NH2

O

1´

1

ribonucleoside ： AR, GR, UR, CR

目 录

目 录

(2) The naming of nucleoside or nucleotides

目 录

目 录

Biological active substances containing nucleotide ： NAD+ 、 NADP+ 、 CoA-SH 、 FAD etc, containing AMP

nucleotides ： NMP ， NDP ， NTP cyclic nucleotide: cAMP ， cGMP

NO

CH2O

OHOH

N

NN

NH2

P

O

OH

OH

AMPAMP

NO

CH2O

OHOH

N

NN

NH2

P

O

OH

OP

O

OH

OH

ADPADP

NO

CH2O

OHOH

N

NN

NH2

P

O

OH

OP

O

OH

OP

O

OH

OH

ATPATP

NO

CH2O

OHO

N

NN

NH2

PO

OH

cAMPcAMP

NADP+NAD+

Some important free nucleotide and Some important free nucleotide and their ramifications in organismstheir ramifications in organisms

目 录

5´end

3´end

(3)The linkage of nucleotides

The nucleotides ar

e linked together by pho

sphodiester bonds to fo

rm polynucleotides, na

mely nucleic acids

C

G

A

目 录

5′end

3′end

C

G

A

2. Primary Structure of nucleic acids

Definition The linear sequence of (deoxy)nucleotides ( or base sequences ) in DNA (RNA) is termed primary structure of DNA (RNA).Linkage bondphosphodiester linkage

A G

P5 P

T

P

G

P

C

P

T

P OH 3

writing

5 pApCpTpGpCpT-OH 3

5 A C T G C T 3 目 录

目 录

Tautomerism between the lactam and lactim

1.2 Properties of nucleosides and nucleotides

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

3′end

C

G

A

1. Primary Structure of nucleic acids

Definition The linear sequence of deoxynucleotides ( or base sequences ) in DNA is termed primary structure of DNA.Linkage bondphosphodiester linkage

Backbone: ---Pi-dR-Pi-dR---

目 录

2. Secondary Structure of DNA----Double Helix Model

2.1 Background

目 录

The analysis of base composition of DNAChargaff rules

The analysis of chemical and physical da

ta on DNA building model

Primarily x-ray diffraction data collected by Rosalind franklin and Maurice Wilkins

目 录

Chargaff rulesa. 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 are organized in an antiparallel arrangement (i. e. The two strands run in opposite directions, one strand is oriented 5’3’ and the other is oriented 3’ 5’ ).

3’

5’

3’5’

minor groove

majorgroove

目 录

目 录

(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

3’

5’

3’5’

minor groove

majorgroove

2.2 The key points of DNA Double Helix Model

目 录

Base pairing rules

TA GC

A=T G≡C

Hydrogen bond Hydrogen bond

(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.

目 录

3’

5’

3’5’

minor groove

majorgroove

2.2 The key points of DNA Double Helix Model

目 录

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

(5) Structure stable depends on the vice-bonds

(6) There are minor grooves and major grooves

hydrogen bonds, base stack force

目 录

2.4 Po

lymo

rph

ism o

f seco

nd

ary structu

re of

DN

A

目 录

Forms of DNA

Form Pitch(nm) Residues inclination of base per turn pair from horizontal

A 2.8 11 20°

B 3.4 10 0°

Z 4.5 12 7°

RNA-DNA 2.8 11 20° hybridSource: From Davidson, The biochemistry of the Nucleic Acids, 8thed., revised by Adams, et al. Copyright ©Chapman & Hall, London.

目 录

（ 1 ） Superhelix structure of DNA in prok

aryote ------Circular double stranded superh

elical DNA

2.5 The Superhelix Structure of DNA

目 录

The nucleosome in eukarytic cells

DNA double helix superhelix

nucleosome chromatin chromoso

me

（ 2 ） Superhelix structure of DNA in eukar

yote ------Nucleosomes

The nucleosomes in eukarytic cells

consist of DNA and proteins

目 录

The elementary unit of chromosome is

nucleosome which consists of DNA an

d histone. Histone includes H1, H2A, H

2B, H3, H4. Two molecules of each H2

A, H2B, H3, H4 constitute the core parti

cle of nucleosome.

目 录

Core particles

Linkage DNAH1, H2A, H2B, H3, H4 histones

目 录

目 录

10 nm fiber

Transverse section

length

wis

e section

30 nm fiber

chromosome

300nm solenoid

chromatin

The folding of nucleosomes, the chromatin condensation, the formation of chromosome

目 录

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.

2.6 Functions of DNA

目 录

DNA functions:

1. Template of replication

2. Template of transcription

3. To accept some mutations

目 录

Structures and Functions of RNA

Section Three

目 录

Structure and Function of RNA

目 录

The primary structures of RNA

belong to single stranded linear

polynucleotide, but contain part

self-complementary pairing, namely

hairpin structures

1. Structure of RNA

Elementary unit of nucleic acid is nucleotideRNA: AMP, GMP, CMP, UMP

目 录

Structure Characters of mRNA:(1) 5’-cap sequence: m’GpppNmThe cap functions are to boost the binding of ribosome with mRNA, and to increase the stability of mRNA.

2. Types and Functions of RNAs

2.1 Messenger RNA

目 录

Cap structure of mRNA 5’-end

Guanine

目 录

Structure Characters of mRNA:(2) 3’-end sequence: poly AThe 3’-end functions are related to increase the stability of mRNA and the lifetime worked as template for translation.

5’ cap structure 3’ poly A

5’ uncoding region 3’ uncoding regioncoding region

hnRNA

(intron)

mRNA

(3) Mature process of mRNA *

(exon)

目 录

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

DNA

mRNA

蛋白

转录

翻译

Prokaryotic

cells

Transcript

translation

protein

细胞质

细胞核

DNA

内含子外显子

转录

转录后剪接转运

mRNA

hnRNA

翻译蛋白

真核细胞

Transcript

Prost-transcript modification

translationprotein

Eukaryotic cells

extron

intron

目 录

(1) Composition of transfer RNA, tRNAIt’s the smallest RNA among RNAs, only consists of 70~90 nucleotides.

2.2 Transfer RNA (tRNA)

tRNAs contain some unusual (modified) bases, such as 7-methylguanine, pseudouridine, dehydrouridine,

目 录

N

NH

NH

N

N

O

CH3

CH3

N

N

NH

N

NHCH2 CH C

CH3

CH3

NH

NH

O

O

H

H

H

H

NH

NH

S

O

N,Ndimethylguanine Dihydrouridine

unusual bases

N6-isopreneadenine4-thiouracil

目 录

The tRNA molecules serve as adapters for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids.

(2) Functions of transfer RNA, tRNA

Two roles:

Activating amino acids

Recognizing codons in mRNA

目 录

◆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.

(3) The structure characters of tRNA

目 录

The cloverleaf pattern of tRNA ( secondary structure)

Amino acid arm

DHU loop

TΨC loop

Anticodon loop

variable loop

目 录

5’

3’

AUG

Tyr

The anticodon at the end of a base-paired stem recognizes the triplet nucleotide or codon of the template mRNA.

5’ 3’UAC mRNAGCA CCA UCGAUGMet

CGU

Ala

5’

GGU 5’

Pro

目 录

The cloverleaf pattern of tRNA ( secondary structure)

目 录

The tertiary structure of tRNA

目 录

(1) rRNA structure

(2) rRNA functionsa. A component of ribosomesb. Ribosomes work as the apparatus of protein synthesis

2.3 Ribosomal RNA (rRNA)

目 录

Eukaryotes

5S rRNA

28S rRNA

5.8S rRNA

18S rRNA

Prokaryotes

5S rRNA

23S rRNA

16S rRNA

(3) rRNA sorts ( dependent on the sedimentation coefficient, S)

Large subunit

small subunit

Large subunit

small subunit

目 录

The components of ribosomeProkaryote ( E coli.) Eukaryote （ mouse live

r ）Small

subunit30S 40S

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 23S5S

2940 nucleotides120 nucleotides

28S5.85S

5S

4718nucleotides160nucleotides120nucleotides

proteins 31 Occupy 30% of total weight

49 Occupy 35% of total weight

目 录

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.

2.4 Other Small Stable RNA

目 录

The major sorts of RNA and their functions

In the nucleus or in the cytoplasm mitochondrion functions

Ribosomal RNA rRNA mt rRNA the components of ribosomes

Messenger RNA mRNA mt mRNA the template for translationTransfer RNA tRNA mt tRNA activating AA and recognizing codons on mRNAheterogeneous nuclear RNA hnRNA the precursor of mRNA

Small nuclear RNA snRNA related to the splicing and

transfering of hnRNASmall nucleolus RNA SnoRNA related to the processing and modifying of rRNA

Small cytoplast RNA scRNA/7SL-RNA the components of signal

discriminator for proteins

located in endoplasmic reticulu

m

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)

The Properties of Nucleic Acid

Section Four

目 录

目 录

1. The general properties of nucleic acids

1.1 Acidic molecules 1.2 Macromolecules 1.3 High viscosity (DNA)1.4 Ultraviolet absorption ( 260 nm)

目 录

Extin

ctio

n

coeffi

cie

nt

Ultraviolet absorbent spectrum of various bases (pH 7.0)

Cytosine

Adenine

Guanine

Thymine Uracil

Wavelength

目 录

2. DNA denaturationDefinitionDouble helix of DNA Single strandThe denaturation factorsheating, chemical reagents, ultraviolet light

目 录

The essence of DNA denaturation is the breaking of hydrogen bonds between the double strands of DNA

Natural DNA Denatural DNA

heating

Cooling slowly

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 )

(1) The determination of the amount of DNA or RNAOD260=1.0 is corresponded to

50μg/ml double stranded DNA40μg/ml single stranded DNA （ or RNA ）20μg/ml oligonucleotides

(2) To estimate the purification of nucleic acid samplesPurified DNA : OD260/OD280 = 1.8

Purified RNA: OD260/OD280 = 2.0

The application of OD260

目 录

For example: the change of ultraviolet absorption spectrum of DNA induced by denaturation

The ultraviolet absorption spectrum of DNA Hyperchromic effect ： the phenomenon of OD260 increase com

panied by DNA denaturation in solution 目 录

Natural state

Denatured state

Ab

sorb

en

t valu

e

Wave length

Heated denaturation of DNAHeated denaturation of DNA

Melting curve: a graph in which absorbance versus temerature is plotted

目 录

Temperature-optical density profile for DNA

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

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

Hybridization double stranded molecules of DNA-DNA

denaturation renaturation

Various source DNA

Renaturation process

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

Double stranded DNA 2

Double stranded DNA

heating

heating

single stranded DNA

single stranded DNAHeterogeneous double stranded DNA

cooling

slowly

Cooling slowly

Adding marked single stranded DNA probes

Forming the marked heterogeneous stranded DNA

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

Nucleasescatalytic RNA and DNA

Section 5

目 录

(1) DefinitionNucleases are the enzymes capable of degrading nucleic acids. (2) CatalogueDNase Those which exhibit specificity for deoxyribonucleic acid are referred to as deoxyribonucleases RNase Those which specifically hydrolyze ribonucleic acids are ribonucleaes.Endonuclease Exonuclease

1. Nucleases

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

There are five classes of ribozymes

Ribozymes are RNA molecules with catalytic activity

★ 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

These special DNA sequences with catalytic capability are called deoxyribozymes.

Some single-stranded DNA molecules are capable of adopting intricate tertiary structures and performing efficient catalysis.

At present, deoxyribozymes are only found in laboratories.

目 录

Genomics and Genomics and the Human Genome the Human Genome

ProjectProject

Section 6Section 6

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

1. Hemoglobin diseases ( 血红蛋白病 )

Disease Cases

2. Horsebean disease ( 蚕豆病 )

3. X-Fragile Syndrome (X- 脆性综合征 )

目 录

♣Types of nucleic acids and their functions

Demands

♣ 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 organisms6. 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 i

ntelligent, 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 m

usic.

Miescher had originally intented to study lymphocytes, he col

lected laudable pus from used bandages in the nearby hospital.

He obtained a precipitate from the cell nuclei solution. Obviousl

y this material must have come from the nucleus, and he theref

ore named it nuclein. Using elementary analysis, one of the few

methods available to characterize an unknown compound. He e

xtracted high-molecular-weight DNA from damaged cells event

ually.

目 录

Oswald T. Avery (1877-1955). Courtesy of The Rockefeller University Archives.

Reich

ard, P

. J. Biol. C

hem

. 2002;277:13355-13362

目 录

O.T. Oswald Theodore Avery (1877 ～ 1955)

Oswald Theodore Avery (1877-1955) was a distinguished bacter

iologist and research physician and one of the founders of immunoch

emistry. He is best known, however, as a discoverer that deoxyribon

ucleic acid (DNA) serves as genetic material. The Oswald T. Avery C

ollection 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 stron

g connection between Dr. Avery's work and his own. The work of Av

ery and his lab, observes Dr. Lederberg, was "the historical platfor

m of modern DNA research" and "betokened the molecular revoluti

on in genetics and biomedical science generally.

目 录

Rosalin

d F

rank

lin（1920-1958

）

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 X-

ray 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: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/).