INTRODUCTION TO MOLECULAR BIOLOGY

Definition
Molecular biology is the study of gene structure and
function at the molecular level

What is a gene?
The question was asked by three groups:

The chemists question: of what (which

cellular chemical) is the gene made?

The geneticists question: what does the gene

do?

The physicists question: what are the

thermodynamics of the gene?
What is DATP ? And how it is assembled ? Deoxyadenosine tri phosphate Used in building block in DNA synthesis

You should be able to draw nucleotide? We know that DNA has a polarity when I say 5 prime what does
The above structure is a nucleotide it mean ? What are characteristics of 5 prime and 3 prime ?
. Only one that still has the phosphate it is the 5 prime and the 3
It consists of a: prime has the free hydroxyl group
• phosphate group
• 5-carbon sugar and
• nitrogenous base
.

What is the enzyme that makes RNA ? RNA polymerase

What is the enzyme that make DNA ? DNA Polymerase

What is polymerization ? A process through which a large number of monomer molecules reach together to form a polymer

Monomer ? Small organic molecule that can form a repeating pattern when covalently bounded to one another
Polymer ? Big molecule that are made up of smaller monomer units that are covalently bounded to one another in a repeating pattern

Difference between purine and pyrimidine ?

-

& ·

- E Purine is 2 and it’s large and contain ( A-G )

Pyrimidine is one and it’s small ( C -T )

- -
·
&
-
Why is it called RNA polymerase ? Because it is an enzyme that polymerase a Ribonucleotide / Enzyme responsible for synthesizing RNA molecules
How many phosphate I’m DNA AND RNA ? 3

Why when numbering we use the word prime ? To distinguish between the carbon on nitrogen is bases and carbon on
sugar. / Nitrogenous bases no prime while carbon atom in sugar has prime.

Phosphate is attached to carbon? 5'

Nitrogen bases attached to carbon? 1'

what is the backbone of DNA? sugar and phosphate

in DNA OH is in carbon number? 3'
in RNA OH is in carbon number? 2' AND 3'

what do you release when you want to increase acidity in DNA ? H+ increases acidity
Bond forms between the hydroxyl group (OH) at C1 of the pentose and the Hydrogen (H) at position
Pyrimidine = ?
Purine = ?
Pyrimidine = H1
Purine = H9
Nucleotides are joined together by 5' and 3' carbon atoms of pentose sugar. The linkage is known as: phosphodiester bond.
In a nucleotide, the nitrogen base🧬is joined to the sugar molecule by Glycosidic bond
In a nucleotide, the phosphate is joined to the sugar molecule by ? phosphodiester bond.

DNA IS A helix what does helix mean ? Term used to describe physical structure of DNA That means it is not straight it has angle of distortion
And remember this create major and minor groove what is a. Minor and what is a major groove ?
1 Two distinct grooves known as the minor and the major grooves form on the opposite sides of the base pairs.
2 The formation of minor grooves takes place where the sugar phosphate backbones are far apart.
3 While the formation of major grooves takes place where the sugar phosphate backbones are close together.
 How many phosphate we have ? These phosphate have names alpha beta and gamma the one that is near
called alpha

Glycosidic bond

OH location on which prime

OH = CARBON 3 PRIME
P = CARBON 5 PRIME

DNA MOLECULES IS NEGATIVE CHARGE BECAUSE OF PHOSPHATE ITS ACIDIC

CHEMICAL COMPONENTS OF THE NUCLEIC ACIDS

Nitrogenous bases

Phosphoric acid

Pentoses
 NUCLEOSIDES

Glycoside bond between the

Nitrogenous base and the
Pentose: Nucleoside.

Bond forms between the

hydroxyl group (OH) at C1 of
the pentose and the
Hydrogen (H) at position H1
(pyrimidine) or H9 (purine).
 LES NUCLEOTIDES

Addition of the phosphate group at position C5’ Nucleotides

DNA molecule is acidic due to the phosphate goup. At physiological pH, the
phosphate dissociate and become negatively charged with the release of an H+.

Starting from the carbone 5’, phosphate are called alpha, beta and gamma.
 In DNA and RNA we have a monophosphate nucleotide which phosphate always remains ? Alpha
O Base 1
Starting from the carbone 5', phosphate are called ? alpha, beta and gamma.
phosphodiester bond form between ?
P - O - P - O - P - O - CH2 .
5

Phosphodiester bonds form between the 3' carbon of one sugar molecule and the 5' carbon of the
OH OH
adjacent sugar molecule in the DNA or RNA chain.
and beta O Base I
gamma gone

this is
gone
5 the only nucleotide that has a free phosphate is in carbon number? 5'
P - O - P - O - P - O - CH2 . the only nucleotide that has a free OH is in sugar carbon number ? 3'
OH OH

O Base 3 In DNA and RNA we have a monophosphate nucleotide which phosphate always remains ? Alpha
5

P - O - P - O - P - O - CH2 3

OH OH

The only nucleotide that has free phosphate it’s the first one in position 5’ Hydrolysis is a common form of a chemical reaction where water is mostly used to
Only one has free OH in 3 prime break down the chemical bonds that exists between a particular substance.

The energy for polymerzation comes from? The hydrolysis of the 2 phosphate

Nucleic acids (DNA and RNA) are formed nucleotides polymerization. This reaction is catalysed by ? polymerases

The bond that form between two nucleotides is called?

phosphodiester bond.
bonds form between carbone at position 3' of one sugar and the carbone at position 5' of the next sugar.
Nucleotide polymerization refers to the process of joining individual nucleotides together to form a polymer, which is the long chain structure of nucleic acids like
DNA and RNA. In this process, nucleotides, which are the building blocks of DNA and RNA, are linked together by chemical bonds called phosphodiester bonds.

"Catalyzed" means that the reaction is facilitated or sped up by a catalyst, which is a substance that increases the rate of a chemical reaction without itself being
consumed in the reaction. In the context of nucleotide polymerization, polymerases act as catalysts, promoting the joining of nucleotides into a polymer by
facilitating the formation of phosphodiester bonds between them.
 PHOSPHODIESTER BONDS

Nucleic acids (DNA and RNA) are formed

nucleotides polymerization. This reaction
is catalysed by polymerases.

The bond that form between two

nucleotides is called phosphodiester
bond.

These bonds form between carbone at

position 3’ of one sugar and the carbone at
position 5’ of the next sugar.

Sugar
Phosphodiester
bond
 Example of simplified DNA extraction procedure

No need of microscope to see DNA. All you need is cooking recipe.

Materiel needed

1 large onion,
Salt Know why we use these
Lemon juice,
alcool 95%,
net

Recipe

1) Cut the onion into large piece, add few milliliter of water and smash using a mixer.

2) Filter the mixture through a net and add to the filtred solution salt and lemon juice. Mix well.

3) Add three volumes of ethanol. Mix gently and put in the freezer to help the precipitation of the
nucleic acid.
Representing the DNA double helix
Protein prefer to bind to? Major groove

How many grooves we have ? We have two distinct grooves minor and major
DNA IS DOUBLE STAND HOLDING TOGETHER BY HYDROGEN BOND

Each molecule of DNA is a double helix formed from two complementary strands of nucleotides held together by ?
hydrogen bonds between G-C and A-T base pairs.

DNA is double helix and has a certain angle of distortion what is the angle ? 9 or 7 degrees

Transcription factor binds only to major groove

Example : WE have Aysha , Nouf , Aysha , Shamsa imagine protein recognizes this sequence and binds in the major groove now the same sequence we will put it in the
minor groove it will not bind it will not recognize it it same sequence

Question Sir asked in class and made us search the Answer ? Why does G-C different not the same ? Slightly difference remember the chromosome centromere and
telomere what does telomere do ? It is a single strand this region is not double strand this is characteristic of telomere that’s why chromosome shrink because when you
replicate you always sacrifice some pieces which means all this double stand so it will be not equivalent

Telomere
⑳ ·
Centromere
 PROPERTIES OF THE DOUBLE HELIX

The 2 DNA strand possess 3 major properties:

- Complementary

- Antiparallele

- Helicoidal
Complementary meaning In DNA, complementary means A pairs with T, and C pairs with G, forming specific base pairs.

Antiparallel meaning
Antiparallel DNA strands ensure stable and accurate base pairing, forming the characteristic double helix structure.

To allow the nitrogen bases to meet

The orientation of the sugar-phosphate backbone in each strand is such that one strand runs in the 5' to 3' direction, while the other runs in the opposite 3' to 5' direction.

This arrangement ensures that the distance between the paired bases remains consistent, contributing to the stability of the double helix and facilitating accurate

replication of genetic information. If the strands were parallel, the base pairing and overall structure of the DNA molecule would not be as stable or functional.

Helicoidal meaning
The DNA double helix is a twisted ladder-like structure composed of two long strands that coil around each other.

The helical structure of DNA is a key feature that allows for the storage and transmission of genetic information. It provides stability to the molecule while allowing for
easy access to the genetic code during processes like DNA replication and transcription.

The energy of the ----------- between complementary strands represents the force that hold the strands together. hydrogen bonds
 COMPLEMENTARY STRANDS
The energy of the hydrogen bonds between complementary strands
represents the force that hold the strands together.
G C

For sterical reasons, purine is facing

pyrimidine base and vice-versa.

Edwin Chargaff ,in 1951, demonstrated

that the 4 nucleotides are not equally
present in the DNA and that the ratio
varies a lot between species.
A T

A+G=C+T
A + G / C + T = 1 in reality it’s not 1 either + 2 or - 2
ANTIPARALLELE POLARITY OF THE
POLYNUCLEOTIDE CHAINE
5’carbon
5’
3’
3’carbon

3’
5’
 HELICOIDAL
In a DNA molecule, the two strands are not parallel, but intertwined with each other. Each strand
looks like a helix. The two strands form a "double helix" structure, which was first discovered by
James D. Watson and Francis Crick in 1953.

The double helix can be found under three different form.

Properties of the helix

Concentration of water 70 - 75% Physiological form because it require Certain region with high g-c
Hybrid RNA-
Form A water Form B content Form Z
90 % of water
DNA

Sens of the helix

Right Right Left Right
rotation

Residus per turn 11 10 12 11

Rotation per
33 deg. 36 deg. –30 deg. 33 deg.
-

residu
Distance
0.255 nm 0.34 nm 0.37 nm 0.255 nm
between bases

step 2.8 nm 3.4 nm 4.5 nm 2.8 nm

 Form A Form B

A form appears when the amount of water in the media is around 75%.
B form appears when the amount of water in the media is around 92%.
Z form is seen in the GC-rich regions
DNA-RNA hybrid has an A form.
 MAJOR AND MINOR GROOVE
IN THE B FORM DNA MOLECULE
The intertwined strands make two grooves of different widths, referred to as the major
groove and the minor groove, which may facilitate binding with specific
proteins.
 HYPERCHROMY
Absorbance at 260 nm of DNA solution increase when the double helix is
denatured into single strand.

Single strand

Absorbance Double strand

220 260 300

DNA's hyperchromic effect means that ssDNA absorbs more
UV than does dsDNA.
The absorbance for single-stranded DNA will be 40% higher than that for double stranded DNA at the same concentration.
 What is hyperchromy ? Absorbance at 260 nm of DNA solution increase when the double helix is denatured into single strand.
When the double helix of DNA is denatured into single strands, the absorbance at 260 nm increases.

In DNA's hyperchromic effect ss or dd absorbe more UV and why ? ssDNA absorbs more UV than does dsDNA.
because ss is more exposed to UV because it does not have nitrogen bases stacked

The absorbance for -----stranded DNA will be 40% higher than that for ---- stranded DNA at the same concentration.
single
double

why double stranded DNA has low UV absorbance ?

a double stranded DNA consists of stacked bases that contribute to the stability and the hypochromicity of the DNA.
Double-stranded DNA has low UV absorbance because the bases in the strands stack together, which reduces their exposure to UV light.

what does spectrophotometer do ? it is a machine that measure the absorption of specific wavelength

nucleotide absorbs waveleghth at ?m260 nm

which part of the nucleotide absorbs at 260 nm ? Nitrogen bases

 TEMPERATURE OF FUSION (Tm)
Does not depend on length depend on composition Mainly depend on g-c
The higher g-c higher Tm why? Because g-c base pair has 3 hydrogen bond

The Tm (temperature of fusion or melting temperature) of a given DNA sequence will correspond to the
temperature at which 50% of the DNA is single strand. The Tm depends of the length, composition of the
DNA molecule and the presence of ions in the media.
 THE Tm IS DEPENDENT ON THE CONTENT
IN GC IN THE DNA MOLECULE

The average GC content of a given DNA molecule can be determined from the
Tm of this molecule.

Tm= 4x[GC] + 2x [AT]

TEMPERATURE OF FUSION (Tm) ?
it is the temp. at which 50% of our DNA is denatures and renatured or temp. at which 50% of DNA is ss and ds

if I take 4 pieces of DNA each with 100 nucleotide will they have the same melting point ?
No it does not depend of the length of DNA but depends on composition ( G-C content )

why higher G-C the higjer the melting point ?

because if G-C it has 3 hydrogen bound so requires more energy to break it so more melting point
however , A-T only has 2 hydrogen bound doesn't requires much energy to break
REASSOCIATION KINETIC

Have Chance to meet identical faster

What is reassociation kinetics?
Reassociation kinetics in DNA refers to the process of double-stranded DNA molecules coming back together after being separated, typically measured by the rate at which
this reassociation occurs.

is reassociation kinetics of protein what happens if you lower temp slowly after raising temp to 95% ?
protein will be completely destroyed it will be denatured and it will not renature again.

is reassociation kinetics of DNA what happensif you lower temp slowly after raising temp to 95% ?
DNA will reach the Tm then when you go lower than TM it will renature basically DNA will denature but then it will renature

why DNA can renature but protein can not ?

because in protein folding requires chapron which is a protein that contribute to the folding of protein and chapron is lost in protein

melting temperature ( Tm ) or temp of fusion depends on ?

G-C content the higher G-C content the higher the temp required to denature the DNA
in our DNA some sequence are midly repeated, highly repeated and little bit repeated

If I take the DNA and cut them into small pieces and chop them then denature the DNA then let the DNA renature slowly (when lowering the DNA slowly DNA renatures
when it goes below the Tm)

which pieces of DNA will renature first ?

the DNA pieces containing highly repeated sequences will renature first. This is because these highly repeated sequences are more abundant in the DNA sample, so they
have more opportunities to find complementary sequences and reassociate with each other compared to the mildly repeated or little bit repeated sequences. Therefore,
they will renature more quickly during the renaturation process
when taking a piece of DNA and start denaturing it will not start opening from the end some pieces will be single strandt first and some pieces will take time why and
what does it depend on?
depends of G-C content more A-T faster it will denature
because A-T requires less energy to denature because of the 2 hydrogen bond between them

The denaturation of DNA depends on the strength of the hydrogen bonds between the bases. Adenine (A) pairs with thymine (T) with two hydrogen bonds, while
guanine (G) pairs with cytosine (C) with three hydrogen bonds. DNA regions with more G-C pairs are generally more stable and take longer to denature compared to
regions with more A-T pairs.

The denaturation of DNA depends on ?

the strength of the hydrogen bonds between the bases. G-C content

---rich region separates first and then the ---- rich region.
A-T
G-C
 PARTIALLY DENATURED DNA OBSERVED UNDER
ELECTRONIC MICROSCOPE

Double strand, G-C rich region

(DNA not yet denatured)

Denatured A-T rich region.

Formation of a single strand
containing bubble

A-T rich region separates first and then the GC rich region.
MOLECULAR HYBRIDIZATION
 NUCLEIC ACID HYBRIDIZATION

DNA replication and transcription requires temporarily separation of the two strands.

The separation is catalyzed by different enzymatic activities. In vitro, such separation can also be
achieved by heating the DNA. The heat energy increases the movement of the molecules which
breaks the hydrogen bonds to the separation of the two strand.

The two strands within a DNA molecule can also be separated chemically by basic solutions such as
NaOH or by solutions containing high concentration of urea or formamide. The separated strand will
adopt a random coiling conformation.

By decreasing progressively the temperature, the double helix will form again. This phenomenon is
called HYBRIDIZATION.
 CREATION OF DNA-RNA HYBRID.

Hybridization can occur either:

1- in solution
2- on solid support : immobilization on membranes, on bacteria colonies, chromosomes
and tissues section…