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9/20/2017

Chromosome The DNA


Structure and Genes: For many decades, the
DNA was thought to be
Structure and merely a structural
element.
function of DNA However, the other
crucial advance made
in the 1940s was the
BRYAN LLOYD P. BRETAÑA
identification of DNA as
Faculty the likely carrier of
Department of Biological Sciences
genetic information.
College of Arts and Sciences
University of Southern Mindanao This breakthrough in our
understanding of cells
came from studies of
With slides from:
DR. GHADA ABOU EL-ELLA
inheritance in bacteria
LECTURER OF BIOCHEMISTRY
FACULTY OF VET. MEDICINE
SOUTH VALLEY UNIVERSITY

Griffith’s Experiments Central Dogma


DNA ---------→ RNA---------→Protein.
This unidirectional flow equation represents the
Central Dogma (fundamental law) of molecular
biology.

This is the mechanism whereby inherited


information is used to create actual objects, namely
enzymes and structural proteins.

An exception to the central dogma is that certain


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

Gene Expression Genetic code


Genes are DNA The alphabet of the
sequences that encode genetic code contains only
proteins (the gene four letters (A,T,G,C).
product)
Gene expression refers A number of experiments
to the process whereby confirmed that the genetic
the information code is written in 3-letter
contained in genes words, each of which
begins to have effects codes for particular amino
in the cell. acid.
DNA encodes and
transmits the genetic
information passed A nucleic acid word (3
down from parents to nucleotide letters) is
offspring. referred to as a codon.

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Nucleic acids Nucleotides


Principle information molecule in the cell.
Nucleotides are the unit structure of
All the genetic codes are carried out on the
nucleic acids.
nucleic acids.
Nucleotides composed of 3 components:
Nucleic acid is a linear polymer of nucleotides
Nitrogenous base (A, C, G, T or U)
Pentose sugar
Phosphate

Nitrogenous bases Types of Nucleic acids


There are 2 types of nucleic acids:
There are 2 types: 1. Deoxy-ribonucleic acid (DNA)
Purines: Pentose Sugar is deoxyribose (no OH at 2’ position)
Bases are Purines (A, G) and Pyrimidine (C, T).
Two ring structure
Adenine (A) and
Guanine (G)
Pyrimidines:
Single ring structure
Cytosine (C) and
Thymine (T) or Uracil
(U).

2. Ribonucleic acid (RNA)


Linear Polymerization of Nucleotides
Pentose Sugar is Ribose.
Nucleic acids are
Bases are Purines (A, G) and Pyrimidines (C, U). formed of nucleotide
polymers.
Nucleotides
polymerize together by
phospho-diester
bonds via
condensation reaction.
The phospho-diester
bond is formed
between:
Hydroxyl (OH) group
of the sugar of one
nucleotide.
Phosphate group of
other nucleotide

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Polymerization of Nucleotides Polymerization of Nucleotides


The formed Nucleic acids are polymers of nucleotides.
polynucleotide chain is
formed of: The nucleotides formed of purine or
Negative (-ve) charged
pyrimedine bases linked to phosphorylated
Sugar-Phosphate sugars (nucleotide back bone).
backbone.
Free 5’ phosphate on one
The bases are linked to the pentose sugar to
end (5’ end) form Nucleoside.
Free 3’ hydroxyl on other The nucleotides contain one phosphate
end (3’ end)
Nitrogenous bases are not
group linked to the 5’ carbon of the
in the backbone nucleoside.
Attached to the backbone Nucleotide = Nucleoside + Phosphate group
Free to pair with
nitrogenous bases of other
polynucleotide chain

The polymerization of Complementary base pairing


nucleotides to form
nucleic acids occur by It is the most important
condensation reaction structural feature of nucleic
acids
by making phospho- It connects bases of one
diester bond between polynucleotide chain
5’ phosphate group of (nucleotide polymer) with
complementary bases of other
one nucleotide and 3’ chain
hydroxyl group of Complementary bases are
another nucleotide. bonded together via:
Double hydrogen bond
Polynucleotide chains between A and T (DNA), A and
are always synthesized U (RNA) (A═T or A═U)
in the 5’ to 3’ direction, Triple H-bond between G and C
with a free nucleotide in both DNA or RNA (G≡C)
being added to the 3’
OH group of a growing
chain.

Significance of DNA structure


DNA is a double stranded
complementary base pairing molecule consists of 2
polynucleotide chains running in
The importance of such complementary opposite directions.
base pairing is that each strand of DNA Both strands are complementary
can act as template to direct the to each other.
synthesis of other strand similar to its The bases are on the inside of the
complementary one. molecules and the 2 chains are
joined together by double H-bond
between A and T and triple H-bond
Thus nucleic acids are uniquely capable between C and G.
of directing their own self replication. The base pairing is very specific
which make the 2 strands
complementary to each other.
The information carried by DNA and So each strand contain all the
RNA direct the synthesis of specific required information for synthesis
proteins which control most cellular (replication) of a new copy to its
activities. complementary.

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2- A-form DNA:
Forms of DNA Less common form of DNA ,
more common in RNA
Right handed helix
1- B-form helix: Each turn contain 11 b.p/turn
Contain 2 different grooves:
It is the most common
Major groove: very deep and
form of DNA in cells. narrow
Right-handed helix Minor groove: very shallow and
wide (binding site for RNA)
Turn every 3.4 nm.
Each turn contain 10 base
pairs (the distance between 3- Z-form DNA:
each 2 successive bases is Radical change of B-form
0.34 nm)
Left handed helix, very extended
Contain 2 grooves;
It is GC rich DNA regions.
Major groove (wide):
provide easy access to The sugar base backbone form
bases Zig-Zag shape
Minor groove (narrow): The B to Z transition of DNA
provide poor access. molecule may play a role in gene
regulation.

Denaturing and Annealing of DNA


Hyperchromicity (melting
The DNA double strands
can denatured if heated profile)
(95ºC) or treated with
chemicals. It is used to monitor the DNA
AT regions denature first (2 H denaturation and annealing.
bonds)
GC regions denature last (3 H It is based on the fact that single
bonds)
stranded (SS) DNA gives higher
DNA denaturation is a absorption (due to the stacking
reversible process, as interactions between the
denatured strands can re-
annealed again if cooled. bases) reading than double
This process can be stranded (DS) at wavelength
monitored using the 260º.
hyperchromicity (melting Using melting profile we can
profile).
differentiate between single
stranded and double stranded
DNA.

Hyperchromicity (melting profile) Melting profile continue…..


Melting profile can be also used to give an idea about the type of
Using melting profile we can differentiate between SS DNA and DS base pair rich areas using the fact that:
DNA A═T rich regions: denatured first (low melting point)
SS
G≡C rich regions: denatured last (higher melting
SS point)
Ab260 SS
AT rich DNA
GC/AT DNA
DS
GC rich DNA
Tm1: Small melting temp.
of AT rich DNA
Tm
Temperature Tm2: higher melting temp. DS
of AT/GC equal DNA

Tm (melting temp.): temp. at which 50% of DS DNA denatured to SS Tm3: Highest melting temp. Tm1 Tm2 Tm3
•Heating of SS DNA: little rise of Ab reading of GC rich DNA
• Heating of DS DNA: high rise of Ab reading

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Melting profile continue….. RNA structure


Melting profile can be also used to give an idea about the type of
base pair rich areas using the fact that:
A═T rich regions: denatured first (low melting point)
It is formed of linear polynucleotide
G≡C rich regions: denatured last (higher melting It is generally single stranded
point) The pentose sugar is Ribose
Uracile (U) replace Thymine (T) in the
pyrimidine bases.

Although RNA is generally single


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

Types of RNA RNA structure


Messenger RNA (mRNA):
Carries genetic information copied from DNA in the form of
a series of 3-base code, each of which specifies a particular RNA is a single stranded
amino acid.
polynucleotide molecule.
Transfer RNA (tRNA):
It is the key that read the code on the mRNA.
Each amino acid has its own tRNA, which binds to it and
carries it to the growing end of a polypeptide chain. It can take 3 levels of structure;
Ribosomal RNA (rRNA): Primary: sequence of nucleotides
Associated with a set of proteins to form the ribosomes.
These complex structures, which physically move along the Secondary: hairpin loops (base pairing)
mRNA molecule, catalyze the assembly of amino acids into
protein chain. Tertiary: motifs and 3D foldings
They also bind tRNAs that have the specific amino acids
according to the code.

DNA Replication
RNA structure
Replication of the DNA molecule is semi-conservative,
which means that each parent strand serves as a
template for a new strand and that the two (2) new
DNA molecules each have one old and one new
strand.

DNA replication requires:


A strand of DNA to serve as a template
Substrates - deoxyribonucleoside triphosphates
(dATP, dGTP, dCTP, dTTP).
DNA polymerase - an enzyme that brings the
substrates to the DNA strand template
A source of chemical energy to drive this synthesis
Transfer RNA (tRNA) structure reaction.

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DNA Replication Step 1 - Unwinding and


Nucleotides are always added to the growing strand Exposing Strands
at the 3' end (end with free -OH group). DNA strands are unwound and opened by enzymes called
HELICASES

The hydroxyl group reacts with the phosphate group


on the 5' C of the deoxyribose so the chain grows Helicases act at specific places called ORIGINS OF REPLICATION

Energy is released when the bound linking 2 of the 3 Synthesis of new DNA strands proceeds in both directions from an
phosphate groups to the deoxyribonucleoside origin of replication resulting in a bubble with REPLICATION FORKS
triphosphate breaks at each growing point.

Remaining phosphate group becomes part of the


sugar-phosphate backbone

Step 2 - Priming the Strand Step 3 - Strand


In order to begin making a new strand, a helper Elongation
DNA Polymerase III catalyses elongation of new
strand called a PRIMER is needed to start the DNA strands in prokaryotes
strand.
Two molecules of DNA polymerase III clamp
DNA polymerase, an enzyme, can then add together at the replication forks, each acting on 1
nucleotides to the 3' end of the primer. of the strands

Primer is a short, single strand of RNA (ribonucleic One strand exposed at its 3' end produces a
acid) and is complimentary to the DNA template daughter strand which elongates from its 5' to 3'
strand. end and is called the LEADING STRAND. This
strand is synthesized continuously and grows
from 5' to 3'.
Primers are formed by enzymes called PRIMASES.

Step 3 - Strand Elongation Step 3 - Strand Elongation


When an Okazaki fragment forms:
The second daughter strand is called the
LAGGING STRAND and is antiparallel to the removes the RNA primer and replaces
DNA polymerase I
leading strand. It’s template is exposed from the it with DNA adjacent to the fragment.
5' to 3' end but it must direct the 5' to 3' synthesis
of the lagging strands, since nucleotides are
added at the 3' end of the chain. leaving 1 bond between adjacent fragments
missing.

The lagging strand is constructed in small, A second enzyme called a DNA LIGASE catalyses the
backward directed bits consisting of
discontinuous sections of 100-200 nucleotides in formation of the final bond.
eukaryotes and 1000-2000 nucleotides in
prokaryotes, called OKAZAKI FRAGMENTS.

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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.