Lecture 4

Structure and function of nucleic acid

Contents

1. Composition of nucleic acid2. Structure and function of DNA 3. Structures and functions of

RNA4. Properties of nucleic acid

Brief history• 1869: isolated DNA from salmon sperm (Friedrich Mie

scher)• 1944: proved DNA is genetic materials (Avery et al.)• 1953: discovered DNA double helix (Watson and Cric

k)• 1968: decoded the genetic codes (Nirenberg)

• 1981: invented DNA sequencing method (Gilbert and Sanger)

• 1987: launched the human genome project • 2001: accomplished the draft map of human genome

Nucleic acid

Deoxyribonucleic acid, DNA

Ribonucleic acid, RNA

•DNA and RNA are polymers of nucleotide units.

• DNA (RNA) consists of 4 kinds of ribonucleotide units linked together through covalent bonds. • Each nucleotide unit is composed of

a nitrogenous base a pentose sugar a phosphate group

1. The components of DNA and RNA

1.1 Bases

• Purines : – Adenine (A) – Guanine (G)

• Pyrimidines :– Cytosine (C)– Uracil (U)– Thymine (T)

Thymine (T) is a 5-methyluracil (U)

DNA: A,G,C,TRNA: A,G,C,U

1.2 Ribose (in RNA) and deoxyribose (in DNA) •

• Ribose and deoxyribose predominantly exist in the cyclic form.

•The bases are covalently attached to the 1’ position of a pentose sugar ring, to form a nucleoside

Glycosidic bond

R Ribose or 2’-deoxyribose

1.3 Nucleosides =ribose/deoxyribose + bases

1

Adenosine, guanosine, cytidine, thymidine, uridine

•A nucleotide is a nucleoside with one or more phosphate groups bound covalently to the 3’-, 5’, or ( in ribonucleotides only) the 2’-position. In the case of 5’-position, up to three phosphates may be attached.

Deoxynucleotides (containing deoxyribose)

Ribonucleotides (containing ribose)

Phosphate ester bonds

1.4 Nucleotides = nucleoside + phosphate

BASES NUCLEOSIDES NUCLEOTIDES

Adenine (A)

Adenosine Adenosine 5’-triphosphate (ATP)

Deoxyadenosine

Deoxyadenosine 5’-triphosphate (dATP)

Guanine (G)

Guanosine Guanosine 5’-triphosphate (GTP)

Deoxyguanosine

Deoxy-guanosine 5’-triphosphate (dGTP)

Cytosine (C)

Cytidine Cytidine 5’-triphosphate (CTP)Deoxycytidine Deoxy-cytidine 5’-triphosphate (d

CTP)Uracil (U) Uridine Uridine 5’-triphosphate (UTP)

Thymine (T)

Thymidine/Deoxythymidie

Thymidine/deoxythymidie 5’-triphosphate (dTTP)

nucleic acid nucleotidesphosphate

nucleosides

pentose

bases

P

O

O

OH

OHO

CH2

OHOH

N

N

NH2

O

Nucleic acid

base ribose

DNA A、 G、 C、 T deoxyribose

RNA A、 G、 C、 U ribose

Composition of DNA and RNA

1.5 Some important nucleotides • dATP, dGTP, dCTP, dUTP

– Raw materials for DNA biosynthesis. • ATP, GTP, CTP, UTP

– Raw materials for RNA biosynthesis – Energy donor– Important co-enzymes

• Cycling nucleotides—cAMP, cGMP – Secondary messengers in hormones action.

Nucleic acid derivatives

Multiple phosphate nucleotides adenosine monophosphate (AMP)adenosine diphosphate (ADP)adenosine triphosphate (ATP)

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

2.1 Primary structure Definition: the base sequence (or the nucle

otide sequence) in polydeoxynucleotide chain.

The smallest DNA in nature is virus DNA. The length of φX174 virus DNA is 5,386 bases (a single chain).

The DNA length of human genome is 3,000,000,000 pair bases.

2. Structure and function of DNA

• 3’,5’ phosphodiester bond link nucleotides together to form polynucleotide chains

5’end

3’ end: free hydroxyl (-OH) group

Phosphodiester bond

The structure of a DNA chain can be concisely represented

• An even more abbreviated notation for this chain is – pApCpGpTpA– pACGTA

• The base chain is written in the 5’ →3’ direction

2.2 Secondary structure

The secondary structure is defined as the relative spatial position of all the atoms of nucleotide residues.

•Watson and Crick ,

1953

•The genetic material

of all organisms

except for some

viruses.

•The foundation of the

molecular biology.James D. Watson

Francis H.C. Crick

Secondary structure

— DNA double helix structure

The discovery of DNA double helix

• Chargaff's Rule (A=T, G=C in DNA)

• Franklin, Wilkins: X-ray Diffraction

Refined Structure

•Two separate strands•Antiparellel (5’3’ direction)•Base pairing: hydrogen bonding that holds two strands together•Complementary (sequence)

Essential for replicating DNA and transcribing RNA

5’

3’

3’

5’

• Sugar-phosphate backbones (negatively charged): outside• Base pairs (stack one above the other): inside

DNA double helix

Base pairing

A:T G:C

1234

897 65

4 3 21

B form of DNA double helix • Right-handed helix;•The diameter of the double helix： 2 nm• The distance between two base pairs: 0.34 nm;• Each turn of the helix involves 10 bases pairs, 3.4 nm.

Stable configuration can be maintained by hydrogen bond and base stacking force (hydrophobic interaction).

Groove binding

• Small molecules like drugs bind in the minor groove, whereas particular protein motifs can interact with the major grooves.

• Watson, Crick, and Wilkins shared the Nobel Prize in medicine or physiology in 1962 for this brilliant accomplishment.

• The discovery of the DNA double helix revolutionized biology: it led the way to an understanding of gene function in molecular terms (their work is recognized to mark the beginning of molecular biology).

Conformational variation in double-helical structure

• B-DNA • A-DNA • Z-DNA

• B-form: the duplex structure proposed by Watson and Crick is referred as the B-form DNA.

•It is the standard structure for DNA molecules.

•A-form: at low humidity the DNA molecule will take the A-form:

•The A-form helix is wider and shorter, with a shorter more compact helical structure, than the B-form helix.

• Z-form: the Z-form DNA is adopted by short oligonucleotides. •It is a left-handed double helix in which backbone phosphates zigzag.

2.3 Tertiary structure : • Supercoils: double-stranded circular DNA for

m supercoils if the strands are underwound (negatively supercoiled) or overwound (positively supercoiled).

Relaxed supercoiled

Increasing degree of supercoiling

• If the strands are overwound, form positively supercoiled;

• If the strands are underwound, form negatively supercoiled.

• The DNA in a prokaryotic cell is a supercoil.

• Supercoiling makes the DNA molecule more compact thus important for its packaging in cells.

2.4 Eukaryotic DNA

• DNA in eukaryotic cells is highly packed.

• DNA appears in a highly ordered form called chromosomes during metaphase, whereas shows a relatively loose form of chromatin in other phases.

• The basic unit of chromatin is nucleosome.

• Nucleosomes are composed of DNA and histone proteins.

Nucleosome

• The chromosomal DNA is complexed with five types of histone.

•H1, H2A, H2B, H3 and H4.•Histons are very basic proteins, rich in Arginine and Lysine.

•Nucleosomes: regular association of DNA with histones to form a structure effectively compacting DNA. ”beads”

Beads on a string

• 146 bp of negatively supercoiled DNA winds 1 ¾ turns around a histone octomer.

• H1 histone binds to the DNA spacer.

The importance of packing of DNA into chromosomes

Chromosome is a compact form of the DNA that readily fits inside the cell

To protect DNA from damageDNA in a chromosome can be

transmitted efficiently to both daughter cells during cell division

Chromosome confers an overall organization to each molecule of DNA, which facilitates gene expression as well as recombination.

2.5 Functions of DNA

• The carrier of genetic information.• The template strand involved in

replication and transcription.

Gene: the minimum functional unit in DNA Genome: the total genes in a living cell or living beings.

3. Structures and functions of RNA

Conformational variability of RNA is important for the much more diverse roles of RNA in the cell, when compared to DNA.

Types : • mRNA: messenger RNA, the carrier of genetic infor

mation from DNA to translate into protein • tRNA: transfer RNA , to transport amino acid to rib

osomes to synthesize protein • rRNA: ribosomal RNA, the components of ribosom

es • hnRNA: Heterogeneous nuclear RNA• snRNA: small nuclear RNA

Classes of eukaryotic RNAs

RNA structure

• RNA molecules are largely single-stranded but there are double-stranded regions.

3.1 Messenger RNA( mRNA)

• Function: the carrier of genetic information from DNA for the synthesis of protein.

• Comprises only about 5% of the RNA in the cell.

• Composition: vary considerably in size (500-6000 bases in E. coli)

Eukaryotic mRNA Structure

(1) Capping: linkage of 7-methylguanosine to the 5’ terminal residue.

(2) Tailing: attachment of an adennylate polymer (poly A, 20~250 nucleotides) at the 3’ terminal.

3.2 Transfer RNA (tRNA)

• Primary Structure : – 74~95 bases, the smallest of the three major RNA.

– Modified bases: pseudouridine (ψ) methylguanosine dihydrouridine (D)– The sequence CCA at the 3’ terminus

• They make up 15% of the RNA in the cell. • Function: Transport amino acids to ribosomes for assembly into proteins.• There are at least 20 types of tRNA in one cell.

Secondary structure: cloverleaf

• Four loops and four arms

– Amino acid arm (7bp): to bide amino acid

– D loop(8-14bp) and D arm(3-4bp):

– Anticoden loop(5bp) and arm(7bp): to recognize amino acid coden on the mRNA.

– TψC loop （ 7bp ） and arm(5bp)

– Variable loop(4-5bp or 13-21bp)

•Tertiary structure of tRNA

* The species of rRNA

•Eukaryotes•5S rRNA•28S rRNA•18S rRNA•5.8S rRNA

•Prokaryotes•5S rRNA•23S rRNA•16S rRNA

• S represents Svedberg units, they represent measures of sedimentation rate.

3.3 Ribosomal RNA (rRNA)

• Components of ribosomes. • They make up 80% of the RNA in the cell.

The proposed secondary structure for E.coli 16S rRNA

Ribosomes

• Ribosomes are cytoplasmic structures that synthesize protein, composed of RNA (2/3) and protein (1/3).

• The ribosomes of prokaryotes and eukaryotes are similar in shape and function. The difference between them is the size and chemical composition.

Three Three rRNA5252 proteins

Four Four rRNA8383 proteins

• Ribosomes are ribonucleoprotein particles for synthesizing proteins.

Other RNAs• Small nuclear RNA (snRNA)

– Involved in mRNA processing• Small nucleolar RNA (snoRNA)

– Play a key role in the processing of rRNA molecules

• Small cytoplasmic RNA (scRNA)– Involved in the selection of proteins for export

• Catalytic RNA or Ribozyme• Small interfering RNA (siRNA)

– Interfere with the expression of a specific gene• RNomics

4. Physical and Chemical Properties of Nucleic Acids

General properties• Acidity

– Amphiphilic molecules; normally acidic because of phosphate.

• Viscosity– Solid DNA: white fiber; RNA: white powder. Insol

uble in organic solvents, can be precipitate by ethanol.

• Optical absorption– UV absorption due to aromatic groups.

• Thermal stability– Disassociation of dsDNA (double-stranded DNA) in

to two ssDNAs (single-stranded DNA).

4.1 UV Absorption

• Specific absorption at 260nm. • This can be used to identify

nucleic acid.

The UV absorption spectra of the common ribonucleotides

4.2 Denaturation

• Concept: • The course of hydrogen bonds

broken, 3-D structure was destroyed, the double helix changed into single strand irregular coil.

• Results: (1)the value of 260nm absorption is

increased; (2)biological functions are lost.

• Heat denaturation and Tm

• When DNA were heated to certain temperature, the absorption value at 260nm would increased sharply，which indicates that the double strand helix DNA was separated into single strand.

•Tm (melting temperature of DNA):• The temperature of UV absorption increase to an half of maximum value in DNA denaturation.

• Factors affect Tm: G-C content:

Higher G+C

Less G+C

Temperature

Tm of two DNA molecules with different G+C content

•There are three hydrogen bonds between G-C pair. The more G-C content, the higher Tm value.

4.3 Renaturation of DNA

• When slowly cooling down (Annealing) the denatured DNA solution, the single strand DNA can reform a double strands helix to recover its biological functions.

Molecule hybridization

• During the course of lowing down denaturing temperature, between different resource DNAs or single stand DNA and RNA with complementary bases will repair into a double strands to form a hybrid DNA or DNA-RNA . This course is called molecule hybridization.

Points• The components of DNA and RNA

– Nucleotide: base (A,G,C,T,U), pentose sugar (Ribose and deoxyribose), phosphate group

• Structure and function of DNA– Primary structure: 3’,5’ phosphodiester bond– Secondary structure: DNA double helix– Tertiary structure: supercoil– Eukaryotic chromosomes: nucleosome

• Structures and functions of RNA– mRNA, tRNA, rRNA

• Properties of nucleic acid– UV absorption, denaturation and renaturation, mo

lecule hybridization