Structure and function of Structure and function of DNADNA

Dr. Ghada Abou El-EllaDr. Ghada Abou El-EllaLecturer of biochemistryLecturer of biochemistryFaculty of Vet. MedicineFaculty of Vet. MedicineSouth Valley UniversitySouth Valley University

Central Dogma

DNA ---------→ RNA---------→Protein.DNA ---------→ RNA---------→Protein. This unidirectional flow equation represents the This unidirectional flow equation represents the

Central Dogma Central Dogma (fundamental law)(fundamental law) of molecular of molecular biology.biology.

This is the mechanism whereby inherited information This is the mechanism whereby inherited information is used to create actual objects, namely enzymes and is used to create actual objects, namely enzymes and structural proteins.structural proteins.

An exception to the central dogma is that certain An exception to the central dogma is that certain

viruses (retroviruses) make DNA from RNA using the viruses (retroviruses) make DNA from RNA using the enzyme reverse transcriptase.enzyme reverse transcriptase.

Gene Gene Expression Genes are DNA sequences that encode Genes are DNA sequences that encode

proteins (the gene product) proteins (the gene product)

Gene expression refers to the process Gene expression refers to the process whereby the information contained in genes whereby the information contained in genes begins to have effects in the cell.begins to have effects in the cell.

DNA encodes and transmits the genetic DNA encodes and transmits the genetic information passed down from parents to information passed down from parents to offspring.offspring.

Genetic code Genetic code The alphabet of the genetic code contains The alphabet of the genetic code contains

only four letters (A,T,G,C).only four letters (A,T,G,C).

A number of experiments confirmed that the A number of experiments confirmed that the genetic code is written in 3-letter words, each genetic code is written in 3-letter words, each of which codes for particular amino acid.of which codes for particular amino acid.

A nucleic acid word (3 nucleotide letters) is A nucleic acid word (3 nucleotide letters) is referred to as a referred to as a codon.codon.

Nucleic acidsPrinciple information molecule in the Principle information molecule in the

cell.cell.

All the genetic codes are carried out on All the genetic codes are carried out on the nucleic acids.the nucleic acids.

Nucleic acid is a linear polymer of Nucleic acid is a linear polymer of nucleotidesnucleotides

NucleotidesNucleotides are the unit structure of Nucleotides are the unit structure of

nucleic acids.nucleic acids.Nucleotides composed of 3 Nucleotides composed of 3

components:components: Nitrogenous base (A, C, G, T or U)Nitrogenous base (A, C, G, T or U) Pentose sugarPentose sugar PhosphatePhosphate

Nitrogenous basesThere are 2 types:There are 2 types:

Purines:Purines:Two ring structureTwo ring structureAdenine (A) and Guanine (G)Adenine (A) and Guanine (G)

Pyrimidines:Pyrimidines:Single ring structureSingle ring structureCytosine (C) and Thymine (T) or Uracil (U). Cytosine (C) and Thymine (T) or Uracil (U).

Nucleotide bases

Types of Nucleic acidsThere are 2 types of nucleic acids:There are 2 types of nucleic acids:

1.1. Deoxy-ribonucleic acidDeoxy-ribonucleic acid (DNA) (DNA) Pentose Sugar is deoxyribose (no OH at 2’ position) Pentose Sugar is deoxyribose (no OH at 2’ position) Bases are Purines (A, G) and Pyrimidine (C, T).Bases are Purines (A, G) and Pyrimidine (C, T).

2.2. Ribonucleic acidRibonucleic acid (RNA) (RNA) Pentose Sugar is Ribose.Pentose Sugar is Ribose. Bases are Purines (A, G) and Pyrimidines (C, U).Bases are Purines (A, G) and Pyrimidines (C, U).

Linear Polymerization of Nucleotides Nucleic acids are Nucleic acids are

formed of nucleotide formed of nucleotide polymers.polymers.

Nucleotides Nucleotides polymerize together by polymerize together by phospho-diester phospho-diester bondsbonds via via condensation reaction.condensation reaction.

The phospho-diester The phospho-diester bond is formed bond is formed between:between: Hydroxyl (OH) group Hydroxyl (OH) group

of the sugar of one of the sugar of one nucleotide.nucleotide.

Phosphate group of Phosphate group of other nucleotideother nucleotide

Polymerization of Nucleotides The formed polynucleotide The formed polynucleotide

chain is formed of:chain is formed of: Negative (-ve) charged Negative (-ve) charged

Sugar-Phosphate backbone.Sugar-Phosphate backbone. Free 5’ phosphate on one Free 5’ phosphate on one

end (5’ end)end (5’ end) Free 3’ hydroxyl on other Free 3’ hydroxyl on other

end (3’ end)end (3’ end) Nitrogenous bases are not Nitrogenous bases are not

in the backbonein the backbone Attached to the backboneAttached to the backbone Free to pair with Free to pair with

nitrogenous bases of other nitrogenous bases of other polynucleotide chainpolynucleotide chain

Polymerization of Nucleotides Nucleic acids are polymers of nucleotides.Nucleic acids are polymers of nucleotides. The nucleotides formed of purine or The nucleotides formed of purine or

pyrimedine bases linked to pyrimedine bases linked to phosphorylated phosphorylated sugarssugars (nucleotide back bone). (nucleotide back bone).

The bases are linked to the pentose sugar to The bases are linked to the pentose sugar to form form NucleosideNucleoside..

The nucleotides contain one phosphate The nucleotides contain one phosphate group linked to the 5’ carbon of the group linked to the 5’ carbon of the nucleoside.nucleoside.

Nucleotide = Nucleoside + Phosphate groupNucleotide = Nucleoside + Phosphate group

N.B.N.B. The polymerization of nucleotides to form The polymerization of nucleotides to form

nucleic acids occur by condensation reaction nucleic acids occur by condensation reaction by making phospho-diester bond between 5’ by making phospho-diester bond between 5’ phosphate group of one nucleotide and 3’ phosphate group of one nucleotide and 3’ hydroxyl group of another nucleotide.hydroxyl group of another nucleotide.

Polynucleotide chains are always Polynucleotide chains are always synthesized in the 5’ to 3’ direction, with a synthesized in the 5’ to 3’ direction, with a free nucleotide being added to the 3’ OH free nucleotide being added to the 3’ OH group of a growing chain. group of a growing chain.

Complementary base pairing It is the most important structural feature of It is the most important structural feature of

nucleic acidsnucleic acids It connects bases of one polynucleotide It connects bases of one polynucleotide

chain (nucleotide polymer) with chain (nucleotide polymer) with complementary bases of other chaincomplementary bases of other chain

Complementary bases are bonded together Complementary bases are bonded together via:via: Double hydrogen bond between A and T (DNA), A Double hydrogen bond between A and T (DNA), A

and U (RNA) and U (RNA) (A═T or A═U)(A═T or A═U) Triple H-bond between G and C in both DNA or Triple H-bond between G and C in both DNA or

RNA RNA (G≡C)(G≡C)

Base pairing

Significance of complementary Significance of complementary base pairingbase pairing

The importance of such complementary base The importance of such complementary base pairing is that each strand of DNA can act as pairing is that each strand of DNA can act as template to direct the synthesis of other template to direct the synthesis of other strand similar to its complementary one.strand similar to its complementary one.

Thus Thus nucleic acids are uniquely capable of nucleic acids are uniquely capable of directing their own self replicationdirecting their own self replication..

The information carried by DNA and RNA The information carried by DNA and RNA direct the synthesis of specific proteins direct the synthesis of specific proteins which control most cellular activities.which control most cellular activities.

DNA structureDNA structure DNA is a double stranded molecule consists of 2 DNA is a double stranded molecule consists of 2

polynucleotide chains running in opposite directions.polynucleotide chains running in opposite directions. Both strands are complementary to each other.Both strands are complementary to each other. The bases are on the inside of the molecules and the The bases are on the inside of the molecules and the

2 chains are joined together by double H-bond 2 chains are joined together by double H-bond between A and T and triple H-bond between C and G.between A and T and triple H-bond between C and G.

The base pairing is very specific which make the 2 The base pairing is very specific which make the 2 strands complementary to each other.strands complementary to each other.

So each strand contain all the required information So each strand contain all the required information for synthesis (replication) of a new copy to its for synthesis (replication) of a new copy to its complementary.complementary.

Forms of DNA

1- 1- B-form helixB-form helix::It is the most common form of DNA in It is the most common form of DNA in

cells.cells.Right-handed helixRight-handed helixTurn every 3.4 nm.Turn every 3.4 nm.Each turn contain 10 base pairs (the distance Each turn contain 10 base pairs (the distance

between each 2 successive bases is 0.34 nm)between each 2 successive bases is 0.34 nm)Contain 2 grooves;Contain 2 grooves;

Major groove (wide): provide easy access to basesMajor groove (wide): provide easy access to bases Minor groove (narrow): provide poor access. Minor groove (narrow): provide poor access.

2- 2- A-form DNAA-form DNA:: Less common form of DNA , more common in Less common form of DNA , more common in

RNARNA Right handed helixRight handed helix Each turn contain 11 b.p/turnEach turn contain 11 b.p/turn Contain 2 different grooves:Contain 2 different grooves:

Major groove: very deep and narrowMajor groove: very deep and narrow Minor groove: very shallow and wide (binding site for RNA)Minor groove: very shallow and wide (binding site for RNA)

3-3- Z-form DNA: Radical change of B-form

Left handed helix, very extended It is GC rich DNA regions.The sugar base backbone form Zig-Zag shapeThe B to Z transition of DNA molecule may play a role in

gene regulation.

Denaturing and Annealing of DNA The DNA double strands can denatured if The DNA double strands can denatured if

heated (95ºC) or treated with chemicals.heated (95ºC) or treated with chemicals. AT regions denature first (2 H bonds)AT regions denature first (2 H bonds) GC regions denature last (3 H bonds)GC regions denature last (3 H bonds)

DNA denaturation is a reversible process, as DNA denaturation is a reversible process, as denatured strands can re-annealed again if denatured strands can re-annealed again if cooled.cooled.

This process can be monitored using the This process can be monitored using the hyperchromicity (melting profile).hyperchromicity (melting profile).

Hyperchromicity (melting profile) It is used to monitor the DNA denaturation and It is used to monitor the DNA denaturation and

annealing.annealing.

It is based on the fact that single stranded (SS) It is based on the fact that single stranded (SS) DNA gives higher absorbtion reading than DNA gives higher absorbtion reading than double stranded (DS) at wavelength 260º.double stranded (DS) at wavelength 260º.

Using melting profile we can differentiate Using melting profile we can differentiate between single stranded and double stranded between single stranded and double stranded DNA. DNA.

Hyperchromicity (melting profile)

DS

SS

SSAb260

Tm

Temperature

Tm (melting temp.): temp. at which 50% of DS DNA denatured to SS•Heating of SS DNA: little rise of Ab reading• Heating of DS DNA: high rise of Ab reading

Using melting profile we can differentiate between SS DNA and DS DNA

Melting profile continue…..Melting profile can be also used to give Melting profile can be also used to give

an idea about the type of base pair rich an idea about the type of base pair rich areas using the fact that:areas using the fact that: A═T rich regions: denatured first (low melting point)A═T rich regions: denatured first (low melting point) G≡C rich regions: denatured last (higher melting G≡C rich regions: denatured last (higher melting

point)point)

DS

SS

GC rich DNA

AT rich DNAGC/AT DNA

Tm1 Tm2 Tm3

Tm1: Small melting temp. of AT rich DNA

Tm2: higher melting temp. of AT/GC equal DNA

Tm3: Highest melting temp. of GC rich DNA

RNA structure

It is formed of linear polynucleotideIt is formed of linear polynucleotide It is generally single stranded It is generally single stranded The pentose sugar is RiboseThe pentose sugar is Ribose Uracile (U) replace Thymine (T) in the Uracile (U) replace Thymine (T) in the

pyrimidine bases.pyrimidine bases.

Although RNA is generally single stranded, Although RNA is generally single stranded, intra-molecular H-bond base pairing occur intra-molecular H-bond base pairing occur between complementary bases on the same between complementary bases on the same molecule (secondary structure) molecule (secondary structure)

Types of RNA Messenger RNA (mRNA)Messenger RNA (mRNA)::

Carries genetic information copied from DNA in the form of Carries genetic information copied from DNA in the form of a series of 3-base code, each of which specifies a particular a series of 3-base code, each of which specifies a particular amino acid.amino acid.

Transfer RNA (tRNA)Transfer RNA (tRNA):: It is the key that read the code on the mRNA.It is the key that read the code on the mRNA. Each amino acid has its own tRNA, which binds to it and Each amino acid has its own tRNA, which binds to it and

carries it to the growing end of a polypeptide chain.carries it to the growing end of a polypeptide chain. Ribosomal RNA (rRNA)Ribosomal RNA (rRNA)::

Associated with a set of proteins to form the ribosomes.Associated with a set of proteins to form the ribosomes. These complex structures, which physically move along the These complex structures, which physically move along the

mRNA molecule, catalyze the assembly of amino acids into mRNA molecule, catalyze the assembly of amino acids into protein chain.protein chain.

They also bind tRNAs that have the specific amino acids They also bind tRNAs that have the specific amino acids according to the code.according to the code.

RNA structureRNA is a single stranded RNA is a single stranded

polynucleotide molecule.polynucleotide molecule.

It can take 3 levels of structure;It can take 3 levels of structure;Primary: sequence of nucleotidesPrimary: sequence of nucleotidesSecondary: hairpin loops (base pairing)Secondary: hairpin loops (base pairing)Tertiary: motifs and 3D foldingsTertiary: motifs and 3D foldings

RNA structure

Transfer RNA (tRNA) structure

DNA ReplicationDNA Replication Replication of the DNA molecule is semi-conservative, Replication of the DNA molecule is semi-conservative,

which means that each parent strand serves as a which means that each parent strand serves as a template for a new strand and that the two (2) new template for a new strand and that the two (2) new DNA molecules each have one old and one new DNA molecules each have one old and one new strand. strand.

DNA replication requires: DNA replication requires: A strand of DNA to serve as a A strand of DNA to serve as a template template SubstratesSubstrates - deoxyribonucleoside triphosphates - deoxyribonucleoside triphosphates

(dATP, dGTP, dCTP, dTTP). (dATP, dGTP, dCTP, dTTP). DNA polymeraseDNA polymerase - an enzyme that brings the - an enzyme that brings the

substrates to the DNA strand template substrates to the DNA strand template A source of A source of chemical energychemical energy to drive this synthesis to drive this synthesis

reaction. reaction.

DNA ReplicationDNA Replication Nucleotides are always added to the growing strand Nucleotides are always added to the growing strand

at the 3' end (end with free -OH group). at the 3' end (end with free -OH group).  The hydroxyl group reacts with the phosphate group The hydroxyl group reacts with the phosphate group

on the 5' C of the deoxyribose so the chain grows on the 5' C of the deoxyribose so the chain grows

Energy is released when the bound linking 2 of the 3 Energy is released when the bound linking 2 of the 3 phosphate groups to the deoxyribonucleoside phosphate groups to the deoxyribonucleoside triphosphate breaks triphosphate breaks

Remaining phosphate group becomes part of the Remaining phosphate group becomes part of the sugar-phosphate backbone sugar-phosphate backbone

Step 1 - Unwinding and Exposing Step 1 - Unwinding and Exposing StrandsStrands

DNA strands are unwound and opened by DNA strands are unwound and opened by enzymes called enzymes called HELICASES HELICASES

Helicases act at specific places called Helicases act at specific places called ORIGINS OF REPLICATIONORIGINS OF REPLICATION

Synthesis of new DNA strands proceeds in Synthesis of new DNA strands proceeds in both directions from an origin of replication both directions from an origin of replication resulting in a bubble with resulting in a bubble with REPLICATION REPLICATION FORKSFORKS at each growing point. at each growing point.

Step 2 - Priming the StrandStep 2 - Priming the Strand In order to begin making a new strand, a helper In order to begin making a new strand, a helper

strand called a strand called a PRIMERPRIMER is needed to start the is needed to start the strand. strand.

DNA polymeraseDNA polymerase, an enzyme, can then add , an enzyme, can then add nucleotides to the 3' end of the primer. nucleotides to the 3' end of the primer.

Primer is a short, single strand of RNA (ribonucleic Primer is a short, single strand of RNA (ribonucleic acid) and is complimentary to the DNA template acid) and is complimentary to the DNA template strand.strand.

Primers are formed by enzymes called Primers are formed by enzymes called PRIMASES.PRIMASES.

Step 3 - Strand Elongation Step 3 - Strand Elongation DNA Polymerase IIIDNA Polymerase III catalyses elongation of new catalyses elongation of new

DNA strands in prokaryotes DNA strands in prokaryotes

Two molecules of DNA polymerase III clamp Two molecules of DNA polymerase III clamp together at the replication forks, each acting on 1 together at the replication forks, each acting on 1 of the strands of the strands

One strand exposed at its 3' end produces a One strand exposed at its 3' end produces a daughter strand which elongates from its 5' to 3' daughter strand which elongates from its 5' to 3' end and is called the LEADING STRAND.  This end and is called the LEADING STRAND.  This strand is synthesized continuously and grows strand is synthesized continuously and grows from 5' to 3'. from 5' to 3'.

Step 3 - Strand Elongation Step 3 - Strand Elongation

The second daughter strand is called the The second daughter strand is called the LAGGING STRANDLAGGING STRAND and is antiparallel to the and is antiparallel to the leading strand.  It’s template is exposed from the leading strand.  It’s template is exposed from the 5' to 3' end but it must direct the 5' to 3' synthesis 5' to 3' end but it must direct the 5' to 3' synthesis of the lagging strands, since nucleotides are of the lagging strands, since nucleotides are added at the 3' end of the chain. added at the 3' end of the chain.

The lagging strand is constructed in small, The lagging strand is constructed in small, backward directed bits consisting of backward directed bits consisting of discontinuous sections of 100-200 nucleotides in discontinuous sections of 100-200 nucleotides in eukaryotes and 1000-2000 nucleotides in eukaryotes and 1000-2000 nucleotides in prokaryotes, called prokaryotes, called OKAZAKI FRAGMENTSOKAZAKI FRAGMENTS. .

Step 3 - Strand ElongationStep 3 - Strand Elongation When an When an Okazaki fragmentOkazaki fragment forms: forms: DNA polymerase IDNA polymerase I removes the RNA primer and removes the RNA primer and

replaces it with DNA adjacent to the fragment.replaces it with DNA adjacent to the fragment.

leaving 1 bond between adjacent fragments leaving 1 bond between adjacent fragments missing.missing.

    A second enzyme called a A second enzyme called a DNA LIGASEDNA LIGASE catalyses the formation of the final bond. catalyses the formation of the final bond.

Telomerase Telomerase is a reverse transcriptase that contain

an RNA template, adds nucleotides to the 3’end of the lagging-strand template and thus prevents shortening of lagging strands during replication of linear DNA molecules such as those of eukaryotic chromosomes.