Chapter 3

Structures and Functions of Nucleic Acids

Nucleic acid
A biopolymer composed of nucleotides linked in a linear sequential order through 3,5 phosphodiester bonds

Classification of nucleic acid

Ribonucleic acid (RNA) is composed of ribonucleotides. in nuclei and cytoplasm participate in the gene expression Deoxyribonucleic acid (DNA) is composed of deoxyribonucleotides. 90% in nuclei and the rest in mitochondria store and carry genetic information; determine the genotype of cells

Interesting history
1944: proved DNA is genetic materials (Avery et al.) 1953: discovered DNA double helix (Watson and Crick) 1968: decoded the genetic codes (Nirenberg) 1975: discovered reverse transcriptase (Temin and Baltimore) 1981: invented DNA sequencing method (Gilbert and Sanger) 1985: invented PCR technique (Mullis) 1987: launched the human genome project 1994: HGP in China 2001: accomplished the draft map of human genome

Section 1

Chemical Components of Nucleic Acids

 1.1 Molecular Constituents

Nucleic acid can be hydrolyzed into nucleotides by nucleases. The hydrolyzed nucleic acid has equal quantity of base, pentose and phosphate.

phosphate nucleic acid nucleotides pentose

nucleosides
bases

Base: Purine
NH2 N N

N 7 8 9 NH

5 4

6 3 N

1N 2

NH

Adenine (A)
O N

NH

NH

NH2

Guanine (G)

Base: Pyrimidine
O

6 1 NH

3 2

NH

NH

Uracil (U)
NH2 N
H3C NH O

NH

NH

Cytosine (C)

Thymine (T)

Pentose
HO CH2 5 O OH HO CH2 O OH

4 3
OH

2
OH OH
-D-2-deoxyribose

-D-ribose

Ribonucleoside
NH2 N HO CH2 O N

1
O

glycosidic bond

OH OH

Purine N-9 or pyrimidine N-1 is connected to pentose (or deoxypentose) C-1 through a glycosidic bond.

Ribonucleotide
NH2
phosphoester bond

O HO P OH OH OH O CH2 O N

N O

A nucleoside (or deoxynucleoside) and a phosphoric acid are linked together through the 5-phosphoester bond.

Nomenclature
base nucleoside nucleotide

guanine
cytosine adenine uracil

guanosine
cytidine adenosine uridine

guanosine monophosphate (GMP) cytidine monophosphate (CMP) adenosine monophosphate (AMP) uridine monophosphate (UMP)
(NMP)

Nomenclature
base
guanine
cytosine adenine thymine

nucleoside
deoxyguanosine
deoxycytidine deoxyadenosine deoxythymidine

nucleotide
deoxyguanosine monophosphate (dGMP)
deoxycytidine monophosphate (dCMP) deoxyadenosine monophosphate (dAMP) deoxythymidine monophosphate (dTMP) (dNMP)

Composition of DNA and RNA

Nucleic acid
DNA

base
AGCT AGCU

ribose
deoxyribose

RNA

ribose

Nucleic acid derivatives

Multiple phosphate nucleotides
adenosine monophosphate (AMP) adenosine diphosphate (ADP) adenosine triphosphate (ATP)
O
NH2 N O N N

NH2 N CH2 N O N N

O O P OH O

O P OH OH
NH2

HO

P OH

HO

P OH

CH2

N O

ATP
N

OH

OH

OH

AMP

O HO P OH O

O P OH OH OH O CH2 N O N

ADP

Nucleic acid derivatives

Cyclic ribonucleotide: 3,5-cAMP, 3,5cGMP, used in signal transduction
NH2 N O CH2 N O N N

cAMP
O P OH O OH

Nucleic acid derivatives

Biologically active systems containing ribonucleotide: NAD+, NADP+, CoA-SH

Phosphoester bond formation

The -P atom of the triphosphate group of a dNTP attacks the C-3 OH group of a nucleotide or an existing DNA chain, and forms a 3phosphoester bond.

Nucleic acid chain extension

A nucleic acid chain, having a phosphate group at 5 end and a -OH group at 3 end, can only be extended from the 3 end.

Phosphodiester bonds
Alternative phosphodiester bonds and pentoses constitute the 53 backbone of nucleic acids.

Section 2
Structures and Functions of Nucleic Acids

 2.1 Primary Structure

The primary structure of DNA and RNA is defined as the nucleotide sequence in the 5 3 direction.

Since the difference among nucleotides is the bases, the primary structure of DNA and RNA is actually the base sequence. The nucleotide chain can be as long as thousands and even more, so that the base sequence variations create phenomenal genetic information.

5' P

OH 3'

5' pApCpTpGpCpTpApApC-OH 3'

5' ACTGCTAAC 3'

 2.2 Secondary structure

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

 2.2.a Chargaffs rules

The base composition of DNA generally varies from one species to another. DNA isolated from different tissues of the same species have the same base composition. The base composition of DNA in a given species does not change with its age, nutritional state, and environmental variations. The molarity of A equals to that of T, and the molarity of G is equal to that of C.

Molarity of bases
A G C T A/T G/C G+C Pu/Py

E. coli
Tuberc ulosis

26.0
15.1 31.7

24.9
34.9 18.3

25.2
35.4 17.4

23.9
14.6 32.6

1.09
1.03 0.97

0.99
0.99 1.05

50.1
70.3 35.7

1.04
1.00 1.00

Yeast

Cow
Pig
Human

29.0
29.8 30.4

21.2
20.7 19.9

21.2
20.7 19.9

28.7
29.1 30.1

1.01
1.02 1.01

1.00
1.00 1.00

42.4
41.4 39.8

1.01
1.01 1.01

Historic X-ray diffraction picture

Building a milestone of life

James Watson and Francis Crick proposed a double helix model of DNA in 1953. It symbolized the new era of modern biology.

 2.2.b Double helix of DNA

Two DNA strands coil together around the same axis to form a right-handed double helix (also called duplex). The two strands run in opposite directions, i.e., antiparallel.
There are 10 base pairs or 3.4nm per turn and the diameter of the helix is 2.0nm.

Antiparallel

Backbone and bases

The hydrophilic backbone is on the outside of the duplex, and the bases lie in the inner portion of the duplex.

Base interactions
The two strands of DNA are stabilized by the base interactions.
The bases on one strand are paired with the complementary bases on another strand through H-bonds, namely GC and A=T. The paired bases are nearly planar and perpendicular to helical axis. Two adjacent base pairs have base-stacking interactions to further enhance the stability of the duplex.

Watson-Crick base pair

Watson-Crick base pair

Base-stacking interaction

Major and minor grooves

Groove binding

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

 2.2.c Polymorphisms of DNA

DNA can resume different forms depending upon their chemical microenvironment, such as ionic strength and relative humidity. B-form DNA is the predominant structure in the aqueous environment of the cells.

A-form and Z-form are also native structures found in biological systems.

Structural features of DNAs

Feature
Helix rotation Base pair per turn Pitch Helical diameter Rise per base pair Glycosyl formation

A-DNA
Right-handed 11 2.46nm 2.55nm 0.26nm Anti-

B-DNA
Right-handed 10 3.4nm 2.0nm 0.34nm Anti-

Z-DNA
Left-handed 12 4.56nm 1.84nm 0.37nm Anti- at C, syn- at G -60 per dimer

Rotation between adjacent base pair Relative humidity

33

36

75%

92%

Triplet DNA

Hoogsteen base pair

The third strand is using Hoogsteen Hbonds to pair with bases on the first strand.

G-quartet DNA
The telomere of DNA is a G-righ sequence, such as 5 (TTGGGG)n 3 4 G residues constitute a plane which is stabilized by Hoogsteen H-bonds.
T G T T 5' T G 3' T T G TG T G T T

G-quartet of DNA
Four strands are arranged in either parallel or antiparallel manner.

 2.3 Supercoil Structure

2.3.a Supercoil structure
The two termini of a linear DNA could be joined to form a circular DNA. The circular DNA is supercoiled, and supercoil can be either positive or negative.

Only the supercoiled DNA demonstrate biological activities.

EM image of supercoiled DNA

Circular DNAs in nature, in general, are negatively supercoiled.

 2.3.b Prokaryotic DNA

Most prokaryotic DNAs are supercoiled.

Different regions have different degrees of supercoiled structures.

About 200 nts will have a supercoil on average.

 2.3.c 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
DNA: ~ 200 bps Histone: basic proteins with many Lys and Arg residues H2A (x2), H2B (x2), H3 (x2), H4 (x2)

Beads on a string
146 bp of negatively supercoiled DNA winds 1 turns around a histone octomer. H1 histone binds to the DNA spacer.

The total length of 46 human chromosomes is about 1.7 m, and becomes 200 nm long after 5 times condensation.

 2.4 Functions of DNA

DNA is fundamental to individual life in terms of They are the material basis of life inheritance, providing the template for RNA synthesis. They are the information basis for biological actions, carrying the genetic information.

 DNA is able to replicate itself in a high fidelity to ensure the genetic information transfer from one generation to the next. DNA can be used as a template to synthesize RNA (transcription), and RNA is further used as the template to synthesize proteins (translation).

DNA posses the inherent and the mutant properties to create the diversity and the uniformity of the biological world.

Gene and genome

A gene is defined as a DNA segment that encodes the genetic information required to produce functional biological products. A gene includes coding regions as well as non-coding regions.

Genome is a complete set of genes of a given species.

Section 3
Structures and Functions of RNA

Classification
mRNA (messenger RNA): template for protein synthesis tRNA (transfer RNA): AA carrier rRNA (ribosomal RNA): a component of ribosome for protein synthesis hnRNA (heterogeneous nuclear RNA): precursor of mRNA snRNA (small nuclei RNA): small RNAs for processing and transporting hnRNA

Classes of eukaryotic RNAs

Unique features
RNA is single stranded, in general. RNA has self-complementary intrachain base paring. The double helical regions of RNA are of the A-form. RNA is susceptible to hydrolysis.

 3.1 Messenger RNA

mRNA is the template for protein synthesis, that is, to translate each genetic codon on mRNA into each AA in proteins. Each genetic codon is a set of three continuous nucleotides on mRNA.

mRNAs constitute 5% of total RNAs.

mRNAs vary significantly in life spans. hnRNA is the precursor of mRNA.

mRNA structure
5'-cap
AUG UAA

3'-poly A tail AAA.....AAA

coding region 5' non-coding region 3' non-coding region

mRNA maturation
hnRNA contains introns and exons. Exons are the sequences encoding proteins, and introns are non-coding portions. Splicing process of hnRNA removes introns and makes mRNA become matured. The matured mRNA has special structure features, including 5-cap and 3-poly A tail.

5-cap

OH OH H H H H2N HN O N N N CH3 H 5' O CH2

NH2 N N O O O 5' O P O P O P O CH2 N O N O O O H 2'H 3' H H OCH3 O

-

mRNA chain

5-cap addition

5-cap addition
Methylation can occur at different sites on G or A. 5-cap can be bound with CBP, benefiting transporting from nucleus to cytoplasm.

5-cap can be recognized by translation initiation factor.

It protects the 5-end from exonucleases.

Poly A tail
20-200 adenine nucleotides at 3 end
a un-translated sequence. Related with mRNA degradation that begins with poly A tail shortening. Associate with poly A tail binding proteins for protection

Poly A tailing

hnRNA splicing
intron exon

hnRNA

mRNA

Matured mRNA of eukaryote

5'-cap
AUG UAA

3'-poly A tail AAA.....AAA

coding region 5' non-coding region 3' non-coding region

 3.2 Transfer RNA

tRNA serves as an amino acid carrier to transport AA for protein synthesis. tRNA is about 15% of total RNA. tRNA is 65-100 nucleotides long. There are at least 20 types of tRNA in one cell.

Structure of tRNA
The overall structure is a cloveleaf, reversed L-shape structure. There are three loops (DHU loop, anticodon loop, TC loop), and four stems. The 3-D structure is stabilized by hydrogen bonds of local intrachain base pairs on these stems.

Reversed L-shape structure

Two key sites of tRNA

A tRNA molecule has an amino acid attachment site and a template-recognition site, bridging DNA and protein. The template-recognition site is a sequence of three bases called the anticodon complementary to the mRNA codon.

Codon and anticodon

The anticodon on tRNA pairs with the codon on mRNA.

Amino acid attachment

The OH group at the 3' end of tRNA links covalently to an amino acid.
Only the attached AA becomes activated and capable of being transported.

Rare Bases
tRNA contains a high portion of unusual bases.

 3.3 Ribosomal RNA

rRNA provides a proper place for protein synthesis.

rRNA is the most abundant RNA in cells (>80%).

rRNA assembles with numerous ribosomal proteins to form ribosomes.

Ribosomes
Ribosomes associate with mRNA to form a place for protein synthesis. Ribosomes of eukaryotes and prokaryotes are similar in shapes and functions.

Components of ribosomes
Prokaryote (E.coli)
Smaller subunit rRNA proteins Larger subunit rRNA 30s 16s 21 50s 23s 5s 31

Eukaryote (Liver of mouse)

40s 1542 nucleotides 18s 40% of total weight 33 60s 2940 nucleotides 28s 120 nucleotides 5.85s 5s 30% of total weight 49

1874 nucleotides 50% of total weight

proteins

4718 nucleotides 160nucleotides 120nucleotides 35% of total weight

Ribosome of E. coli

23S rRNA 31 proteins

5S rRNA

50S large subunit

70S ribosome 30S small subunit

16S rRNA 21 proteins

Secondary structure of 18S rRNA

The secondary structure of rRNA has many loops and stems, which can bind ribosomal proteins to form an assembly for protein synthesis.

Ribosomal complex

N E m7GpppG mRNA P A AAA...AAA

Polysomes

5' mRNA

3'

EM of polysomes

Section 4
Physical and Chemical Properties of Nucleic Acids

General properties
Acidity
Negative backbone

Viscosity
Concentration and aggregation effects

Optical absorption
UV absorption due to aromatic groups

Thermal stability
Disassociation of dsDNA (double-stranded DNA) into two ssDNAs (single-stranded DNA)

 4.1 UV Absorption

Application of OD260
Quantify DNAs or RNAs
OD260=1.0 equals to
50g/ml dsDNA 40g/ml ssDNA (or RNA

20g/ml oligonucleotide

Determine the purity of nucleic acid samples

pure DNA: OD260/OD280 = 1.8
pure RNA: OD260/OD280 = 2.0

Transition of dsDNA to ssDNA

The absorbance at 260nm of a DNA solution increases when a dsDNA is melted into two single strands. The change is called hyperchromicity.

Melting curve of dsDNA

DNA melting
Melting curve: a graphic presentation of the absorbance of dsDNA at 260nm versus the temperature. Melting temperature (Tm): the temperature at which the UV adsorption reaches the half of the maximum value, also means that about 50% of the dsDNA is disassociated into the single-stranded DNA.

Melting curve shift

Tm of dsDNA depends on its average G+C content. The higher the G+C content, the higher the Tm.

 4.2 Thermal stability

Dissociation of dsDNA into two ssDNAs is referred to as denaturation. Denaturation can be partially and completely. The nature of the denaturation is the breakage of H-bonds. Denaturation is a common and important process in nature.

Denaturation of DNA

Extremes in pH or high temperature Cooperative unwinding of DNA strands

EM image of denatured DNA

Renaturation of DNA
Two separated complementary DNA strands can rejoin together to form a double helical form spontaneously when the temperature or pH returns to the biological range. This process is called renaturation or annealing.

 4.3 Hybridization
The ability of DNA to melt and anneal reversibly is extremely important.
An association between two different polynucleotide chains whose base sequences are complementary is referred to as hybridization. The stability of the hybridized strand depends on the complementary degree.

Two dsDNA molecules from different species are completely denutured by heating. When mixed and slowly cooled, complementary DNA strands of each species will associate and anneal to form normal duplexes.

 Two ssDNAs, two ssRNAs, as well as one ssDNA and one ssRNA can also be hybridized. Ionic strength, degree of complementary, temperature, as well as base composition, fragment length of nucleic acids will affect the hybridization. It is a common phenomenon in biology, and has been used as a convenient techniques in medicine and biology.

Target DNA detection

complementary hybridization
probe: target: . TAGCTGAG . ATCGACTC

mismatched hybridization
probe: . TAGCTGAG non-target: . ATCAGCTC

Applications
Gene structure and expression
Microarray or gene chip mRNA separation Gene diagnosis and therapy PCR technique

Section 5
Nuclease

Definition and classification

Nucleases are enzymes that are able to hydrolyze phosphoester bonds and cleave DNA or RNA into fragments.
Deoxyribonuclease (DNase) - specially cleave DNA Ribonuclease (RNase) - specially cleave RNA

Classification
Exonucleases They can cleave terminal nucleotides either from 5-end or from 3-end, such as enzymes used in the DNA replication. Endonucleases They can cleave internally at either 3 or 5 side of a phosphate group, such as the restriction endonucleases used to construct the recombinant DNA.

Exonuclease

Endonuclease

Endonuclease

Exonuclease

3 5

Applications
Participate in DNA synthesis and repair, as well as RNA post-translational modification Digest nucleic acids of food for better absorption

Degrade the invaded nucleic acids

Construct the recombinant DNA