molecular genetics

Molecular Genetics
Dr. Reda Gaafar
Associate Professor of Molecular Genetics
and Biotechnology

• To fulfill its role, the genetic material must
meet several criteria
1. Information: It must contain the information
necessary to make an entire organism
2. Transmission: It must be passed from parent to
offspring
3. Replication: It must be replicated accurately
• Progeny cells have the same genetic information as the
parental cell.
4. Variation: It must be capable of changes
• Without change, organisms would be incapable of variation
and adaptation, and evolution could not occur.
Identification of DNA as the Genetic
material
Dr. Reda Gaafar Associate Professor of Plant Molecular Genetics & Biotechnology

History:
Miescher isolated nuclei from pus (white blood cells) in
1869
Found a novel phosphorus-bearing substance = nuclein
Nuclein is mostly chromatin, a complex of DNA and
chromosomal proteins
It was found to be acidic and called nucleic acids
Nucleic acids are of two types DNA (deoxyribonucleic
acid) and RNA (ribonucleic acid)
Feulgen discovered specific stain for DNA: nucleic acid
was found to be localized in nuclei and chromosomes in
contrast to RNA (cytoplasm)
Nature of the genetic material
(DNA is the genetic material)

1. Bacterial Transformation
(is the process of adding a foreign DNA fragment from a donor
genome into genome of a recipient cell)
Griffith studied a bacterium (Diplococcus pneumoniae)
now known as Streptococcus pneumoniae
S. pneumoniae comes in two strains
S  Smooth
Secrete a polysaccharide capsule
Protects bacterium from the immune system of animals
Produce smooth colonies on solid media
R  Rough
Unable to secrete a capsule
Produce colonies with a rough appearance
Evidences that DNA is the
genetic material

 In 1928, Griffith conducted experiments using two strains of
S. pneumoniae: type IIIS and type IIR
1. Inject mouse with live type IIIS bacteria
 Mouse died
 Type IIIS bacteria recovered from the mouse’s blood
2. Inject mouse with live type IIR bacteria
 Mouse survived
 No living bacteria isolated from the mouse’s blood
3. Inject mouse with heat-killed type IIIS bacteria
 Mouse survived
 No living bacteria isolated from the mouse’s blood
4. Inject mouse with live type IIR + heat-killed type IIIS cells
 Mouse died
 Type IIIS bacteria recovered from the mouse’s blood
Evidences that DNA is the genetic material

Outline of Griffith’s Experiments

 Griffith concluded that something from the
dead type IIIS was transforming type IIR
into type IIIS
 He called this process transformation
 The substance that allowed this to happen
was termed the transformation principle
 Griffith did not know what it was
 The nature of the transforming principle was
determined using experimental approaches
that incorporated various biochemical
techniques

DNA is the transforming material
In 1944 Avery, Macleod and McCarty repeated Griffith’s
experiment using purified cell extracts and
discovered:
- removal of all protein from the transforming
material did not destroy its ability to transform R
strain cells
- DNA-digesting enzymes destroyed all
transforming ability
- the transforming material is DNA
genetic material

 Avery et al also conducted the following experiments
 To further verify that DNA, and not a contaminant (RNA
or protein) is the genetic material

2. Bacterial virus (bacteriophage T2)
Hershey-Chase Experiment
In 1952, Alfred Hershey and Marsha Chase provided
further evidence that DNA is the genetic material
 They studied the bacteriophage T2
 It is relatively simple since its composed of
only two macromolecules
 DNA and protein
genetic material

2. Bacterial virus (bacteriophage T2)
Hershey-Chase Experiment
Hershey and Chase experiment can be summarized as such:
Used radioisotopes to distinguish DNA from proteins
32P labels DNA specifically
35S labels protein specifically
Radioactively-labeled phages were used to infect
nonradioactive Escherichia coli cells
After allowing sufficient time for infection to proceed,
the residual phage particles were sheared off the cells
=> Phage ghosts and E. coli cells were separated
Radioactivity was monitored using a scintillation counter
genetic material

genetic material
Bacterial cell
Phage
DNA
Radioactive
protein
Empty
protein shell
Phage
DNA
Radioactivity
(phage protein)
in liquid
Batch 1:
Sulfur (35S)
Radioactive
DNA
Centrifuge
Pellet (bacterial
cells and contents)
Pellet
Radioactivity
(phage DNA)
in pellet
Centrifuge
Batch 2:
Phosphorus (32P)

1. T2 bacteriophage is composed
of DNA and proteins:
2. Set-up two replicates:
• Label DNA with 32P
• Label Protein with 35S
3. Infected E. coli bacteria with
two types of labeled T2
4. 32P is discovered within the
bacteria and progeny phages,
whereas 35S is not found
within the bacteria but
released with phage ghosts.
Hershey-Chase Bacteriophage Experiment - 1953
Alfred Hershey

3. Biochemical evidence that DNA is the
genetic material
Chargaff and his colleagues (1949-1953) analyzed the
composition of DNA from various species by quantitative
chromatographic methods:
Base equivalence in DNA (i.e. the number of adenine bases
was equal to thymine (A = T) and guanine was equal to
cytosine (G = C). A + G = C + T
genetic material

• By 1947, Erwin Chargaff had developed a series of rules based on a
survey of DNA composition in organisms.
– He already knew that DNA was a polymer of nucleotides
consisting of a nitrogenous base, deoxyribose, and a phosphate
group.
– The bases could be adenine (A), thymine (T), guanine (G), or
cytosine (C).
• Chargaff noted that the DNA composition varies from species to
species.
• In any one species, the four bases are found in characteristic, but
not necessarily equal, ratios.
• The number of adenines was approximately equal to the number of
thymines (%T = %A).
• The number of guanines was approximately equal to the number of
cytosines (%G = %C).
– Human DNA is 30.9% adenine, 29.4% thymine, 19.9% guanine and
19.8% cytosine.

Fraenkel-Conrat and his colleagues in 1950’s found that virus (isolated
from infected tobacco plants) consisted of ribonucleoprotein (a
compound of protein and RNA).
Fraenkel-Conrat Experiment
1.Protein and RNA were separated from two tobacco virus strains.
2.Hybrid viruses were reformed by combining the protein of one strain
with the RNA of the other strain.
3.Hybrid viruses were used to infect healthy tobacco plants: the new
viruses had protein coats identical to the original virus from which RNA
had been isolated & symptoms were identical to those of the strain from
which RNA had been isolated to reconstitute the hybrid virus.
4.The same results were obtained with various combinations of nucleic
acid from 4 different strains and of protein from 3 strains.
Genes of some viruses are made
of RNA

The conclusion was obvious that genes of RNA viruses are
made of RNA and not protein

Tobacco Mosaic Virus (TMV) Experiment
Fraenkel-Conrat & Singer - 1957
Demonstrated that RNA is the genetic material of TMV.

Griffith 1928 & Avery 1944:
DNA (not RNA or protein) is transforming agent.
Hershey-Chase 1953:
DNA (not protein) is the genetic material.
Fraenkel-Conrat & Singer 1957:
RNA (not protein) is genetic material of some viruses, but no known
prokaryotes or eukaryotes use RNA as their genetic material.
Alfred Hershey
Nobel Prize in Physiology or Medicine
1969
Conclusions about these early
experiments

Structure of DNA
James D. Watson/Francis H. Crick 1953 proposed the Double Helix Model
based on two sources of information:
1. Base composition studies of Erwin Chargaff (Chargaff’s Rules)
• indicated double-stranded DNA consists of ~50% purines (A,G) and
~50% pyrimidines (T, C)
• amount of A = amount of T and amount of G = amount of C
• %GC content varies from organism to organism
Examples: %A %T %G %C %GC
Homo sapiens 31.0 31.5 19.1 18.4 37.5
Zea mays 25.6 25.3 24.5 24.6 49.1
Drosophila 27.3 27.6 22.5 22.5 45.0
Aythya americana 25.8 25.8 24.2 24.2 48.4
Structure and Replication of DNA

Structure of DNA
James D. Watson/Francis H. Crick 1953 proposed the Double Helix Model based
on two sources of information:
2. X-ray diffraction studies by Rosalind Franklin & Maurice Wilkins
• In this technique, X-rays are diffracted as they
passed through aligned fibers of purified DNA.
• The diffraction pattern can be used to deduce
the three-dimensional shape of molecules.
Conclusion-DNA is a helical structure with
distinctive regularities, 2 nm, 0.34 nm & 3.4 nm.

• The nitrogenous bases are paired in specific
combinations: adenine with thymine and guanine with
cytosine.
• Pairing like nucleotides did not fit the uniform diameter
indicated by the X-ray data.
– A purine-purine pair would be too wide and a
pyrimidine-pyrimidine pairing would be too short.
– Only a pyrimidine-
purine pairing would
produce the 2-nm
diameter indicated
by the X-ray data.

Double Helix Model of DNA: Six main features
1. Two polynucleotide chains wound in a right-handed (clockwise)
double-helix.
2. Nucleotide chains are anti-parallel: 5’  3’
3’  5’
3. Sugar-phosphate backbones are on the outside of the double helix,
and the bases are oriented towards the central axis.
4. Complementary base pairs from opposite strands are bound together
by weak hydrogen bonds.
A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds).
5’-TATTCCGA-3’
3’-ATAAGGCT-5’
5. Base pairs are 0.34 nm apart. One complete turn of the helix
requires 3.4 nm (10 bases/turn).
6. Sugar-phosphate backbones are not equally-spaced, resulting in
major and minor grooves.

• In addition, Watson and Crick determined that chemical
side groups of the nitrogen bases would form hydrogen
bonds, connecting the two strands.
– Based on details of their
structure, adenine would
form two hydrogen bonds
only with thymine and
guanine would form three
hydrogen bonds only with
cytosine.
– This finding explained
Chargaff’s rules.

Type B-DNA
Other DNA forms
include:
A-DNA:
Right-handed double
helix with 11 bases
per turn; shorter
and wider at 2.3 nm
diameter. Exists in
some DNA-protein
complexes.
Z-DNA:
Left-handed double
helix with 12 bases
per turn; longer and
thinner at 1.8 nm
diameter.

Nucleotide = monomers that make up DNA and RNA
Three components
1. Pentose (5-carbon) sugar
DNA = deoxyribose
RNA = ribose
(compare 2’ carbons)
2. Nitrogenous base
Purines (2 rings)
Adenine
Guanine
Pyrimidines (1 ring)
Cytosine
Thymine (DNA)
Uracil (RNA)
3. Phosphate group attached to 5’ carbon

Nucleotides are linked by phosphodiester bonds to form polynucleotides.
Phosphodiester bond
•Covalent bond between the phosphate group (attached to 5’ carbon) of
one nucleotide and the 3’ carbon of the sugar of another nucleotide.
•This bond is very strong, and for this reason DNA is remarkably stable.
DNA can be boiled and even autoclaved without degrading!
•No kidding, you can autoclave a mouse and get good PCR!
5’ and 3’
The ends of the DNA or RNA chain are not the same. One end of the
chain has a 5’ carbon and the other end has a 3’ carbon.

5’ end
3’ end

Type A, B, and Z conformations of DNA
right-handed helix
11 nucleotide pairs / turn
Thicker (2.3 nm)
right-handed helix
10 nucleotide pairs /turn
2 nm
left-handed helix
12 nucleotide pairs / turn
Longer (1.8 nm)

Outline of Hershey and Chase’s Experiment

• DNA replication – DNA synthesis
– Occurs in the nucleus during ___ of the cell
cycle
– Goal is to make an exact copy of the cell’s
DNA
• Put another way -- goal is to duplicate the
chromosomes.
Replication of DNA
and Chromosomes
Replication

Replication of DNA
Matthew Meselson & Franklin Stahl, 1958
investigated the process of DNA replication in Bacteria
& considered 3 possible mechanisms:
– Conservative model (2 double helix: one contained
completely new strands are synthesized from the original
strands)
– Semiconservative model (2 double helix: both contained
one of the original strands and one new strand)
– Dispersive model (2 double helix: both containing
distinct regions of DNA composed of either both original
strands or both new strands)

DNA helix is:
½ old strand
½ new strand DNA helix
is: of
diverse
regions
DNA helix
is: of 2
new
strands
Models of Replication of DNA

DNA is ½ old DNA
½ new DNA
DNA is of diverse regionsDNA is new DNA

Meselson & Stahl Experiment
• Bacterial cells were grown in a heavy isotope of
nitrogen, 15N
all the DNA incorporated 15N
• cells were switched to media containing lighter 14N
• DNA was extracted from the cells at various time
intervals

• The DNA from different time points was analyzed
for ratio of 15N to 14N it contained
• After 1 round of DNA replication, the DNA
consisted of a 14N-15N hybrid molecule
• After 2 rounds of replication, the DNA contained 2
types of molecules:
– half the DNA was 14N-15N hybrid
– half the DNA was composed of 14N

iGenetics Russell 2010

• Meselson and Stahl concluded that the
mechanism of DNA replication is the
semiconservative model.
• Each strand of DNA acts as a template for
the synthesis of a new strand.
Replication of DNA

DNA replication includes 3 steps:
– Initiation – replication begins at an origin
of replication (Ori)
– Elongation – new strands of DNA are
synthesized by DNA polymerases
– Termination – replication is terminated
differently in prokaryotes and eukaryotes
DNA Replication

Molecular mechanism of DNA
Replication
DNA Replication Process:
• DNA Polymerase: add dNTPs to the growing leading
strand and to the growing Okazaki pieces on the lagging
strand.
• Primase: initiate the Okazaki pieces.
• Exonuclease (e.g. Polymerase I): remove the primer.
• Ligase: join the Okazaki pieces.
• Unwinding proteins (Helicases proteins): unwind the
double helix at the replication fork.
• Topoisomerase: relax the tension caused by unwinding
the double helix.
• DNA Binding Proteins (DBP): stabilize single-stranded
DNA
• Gyrase: unwind the double helix ahead of the replication
fork or for initiation of replication (in bacteria).

DNA polymerase III
• Adds nucleotides to the 3’ end of DNA
• Say…synthesizes DNA in the 5’  3’ direction
• It cannot initiate (start) a new DNA strand
DNA polymerase I
• Removes primer sequences and fills in the gaps with
DNA
Other DNA polymerases (Pol II)
• Proofread the DNA and correct mutations
Primase
• Starts synthesis in the 5’  3’ direction
• Makes a primer sequence to which DNA polymerase
III can add DNA
DNA ligase
• Joins newly made DNA segments after the primer
sequences have been removed
Molecular mechanism of DNA
Replication (Prokaryotes)

Prokaryotic DNA Replication
Bidirectional replication of circular DNA molecules
The chromosome of a prokaryote is a circular molecule
of DNA.
Replication begins at one origin of replication and
proceeds in both directions around the chromosome.

Leading-strand and lagging-strand

Bidirectional replication of circular DNA molecules

The double helix is unwound by the enzyme
helicase
DNA polymerase III (pol III) is the main
polymerase responsible for the majority of
DNA synthesis
DNA polymerase III adds nucleotides to the
3’ end of the daughter strand of DNA

DNA replication is semidiscontinuous.
– pol III can only add nucleotides to the 3’ end of the
newly synthesized strand
– DNA strands are antiparallel to each other
leading strand is synthesized continuously (in the same
direction as the replication fork)
lagging strand is synthesized discontinuously creating
Okazaki fragments

Semidiscontinuous DNA Replication

Joining of Okazaki
fragments

Action of DNA ligase in sealing the nick between
adjacent DNA fragments

Replisome
• The enzymes for DNA replication are contained
within the replisome.
• The replisome consists of
– the primosome - composed of primase and
helicase
– 2 DNA polymerase III molecules
– DNA Polymerase I
– Ligase
• The replication fork moves in 1 direction,
synthesizing both strands simultaneously.

Model for Replisome (replication fork)

Model for the replisome

Eukaryotic DNA Replication
• The larger size and complex packaging of eukaryotic
chromosomes means they must be replicated from multiple
origins of replication.
• The enzymes of eukaryotic DNA replication are more
complex than those of prokaryotic cells.
• In prokaryotes, DNA replication in eukaryotes requires 3
different types of DNA polymerases:
• α Polymerase (alpha pol.) : Pol α (initiation of replication
at origins and for the priming of Okazaki fragments (with
primase) during the discontinuous synthesis of the
lagging strand)

δ Polymerase (delta pol.): Pol δ (extend RNA - DNA
primer chains & completes the replication of the
lagging strand)
ε Polymerase (epsilon pol.) : Pol ε (catalyzes the
replication of the leading strand)
Temporal ordering of DNA replication initiation events in
replication units of eukaryotic chromosomes

Prokaryotes (e.g. E. coli):
• DNA is circular molecule with one origin of replication.
• Replisome:
• Replication fork moves faster (~6000 bases per minute).
It is, therefore, necessary that in eukaryotes
replication be initiated at several points of origin.
Eukaryotes:
• DNA is a long linear molecule with several origins of
replication.
• Replisome
• Replicating fork moves at a slower speed (~ 2600 bases
per minute)
Prokaryotic versus Eukaryotic DNA
Replication

Synthesizing the ends of the
chromosomes is difficult
because of the lack of a
primer.
With each round of DNA
replication, the linear
eukaryotic chromosome
becomes shorter

Choose the correct answers
A) How is it demonstrated that DNA is the “transforming principle”?
1) isolate and study chromosome structure
2) X-ray diffraction of DNA structure
3) treat extracts of type IIIS with different enzymes to look for loss of transforming factor
4) inject mouse with two types of Streptococcus cells and look for death
5) study T2 phage structure
B) Who determined the structure of DNA is a double helix?
1) Miescher 2) Griffith 3) Avery, MacLeod, and McCarty
4) Watson and Crick 5) Hershey and Chase
C) What does the lagging strand only have?
1) DNA polyermase III 2) origin of replication
3) Primer 4) activity of DNA polyermase
5) Okazaki fragments
D) Which of the following is true of eukaryotic DNA replication?
1) histones are synthesized during S phase for chromatin packaging
2) the cell must duplicate the nucleosome structure on the chromosome after replication
3) nucleosomes are dissembled right before replicating a section of DNA
4) eukaryotes have many more DNA polymerases than prokaryotes do
5) all of these are true

Relationship between DNA and protein
• Sequence of nucleotides determines exactly the
sequence of amino acids in proteins
Central Dogma of molecular biology
Crick: One-way sequential flow of information
from DNA to RNA to protein.
Gene Expression: Transcription and
Translation

Central Dogma of molecular biology
Central Dogma: relationship of information
transfer between DNA and protein through
RNA-intermediate
Translation
DNA
ProteinsRNA
Translation
Single-stranded DNA can specify protein

Translation
Two-step process

Transcription
is the synthesis of an RNA copy of a segment
of DNA.
• Only one of the two DNA strands is transcribed into an
RNA
Gene Expression: Transcription

1. mRNA (messenger RNA): mRNAs are the
transcripts of protein-coding genes.
Translation of an mRNA produces a
polypeptide.
2. rRNA (ribosomal RNA): with ribosomal
proteins, makes up the ribosomes—the
structures on which mRNA is translated.
3. tRNA (transfer RNA): brings amino acids to
ribosomes during translation.
4. snRNA (small nuclear RNA): with proteins,
forms complexes that are used in eukaryotic
RNA processing to produce functional
mRNAs.
Types of RNA molecules

Chemical synthesis of RNA transcript is similar to
that of DNA but there are the following differences:
1.Only one strand of DNA is used as a template for RNA
synthesis
2.differ from DNA precursors in having a ribose sugar
and uracil, instead of deoxyribose and thymine
3.RNA molecule is complementary to DNA sequence.
4.Transcription uses RNA polymerase rather than DNA
polymerase
5.Nucleotides are added only to the 3′-OH end of the
growing chain
6.RNA polymerase (unlike DNA polymerase) does not
require a preexisting primer strand to initiate chain
growth

DNA and RNA Differences

RNA synthesis process
•RNA polymerases bind to specific nucleotide sequences
 called promoters
•RNA polymerases initiate the synthesis of RNA
molecules with the help of proteins called transcription
factors
Transcription bubble (unwound segment of DNA) produced by RNA polymerase

Transcription in prokaryotes
Process of transcription can be divided into three
stages:
1. Initiation of a new RNA chain,
2. Elongation of the chain,
3. Termination of transcription and release of the
nascent RNA molecule.
• Single RNA polymerase carries out all transcription
in most prokaryotes,
• Three-five different RNA polymerases are present
in eukaryotes
• Each polymerase responsible for the synthesis
of a distinct class of RNAs

In E. coli, RNA polymerase is complex enzyme which is
made up of 5 subunits (α2, β, β′, σ polypeptides).
• α2, β, and β′ form the tetrameric core enzyme
• σ (sigma factor) is weakly attached.
•Sigma factor recognizes start sequences in the
promoter region of DNA.
•Chain-elongation is catalyzed by the core ((α2, β, β′)
enzyme
•In eukaryotes,three-five RNA polymerases are
present in RNA polymerases I, RNA polymerases II,
RNA polymerases III
• RNA polymerases IV and V: (identified only in
plants and fungi)

In Eukaryotes, RNA polymerases are:
RNA polymerase I: is located in the nucleolus. RNA
polymerase I synthesis of all ribosomal RNAs.
RNA polymerase II: transcribes nuclear genes that
encode proteins.
RNA polymerase III: synthesis of the transfer RNA
molecules, 5S rRNA molecules, and small nuclear RNAs
(snRNAs).
RNA polymerases IV and V:
play important roles in turning off the transcription of
genes by modifying the structure of chromosomes, a
process called chromatin remodeling

All eukaryotic RNA polymerases require the assistance
of proteins called transcription factors to initiate the
synthesis of RNA chains.
Bacterial gene may be divided into three sequences with respect
to its transcription:
1. Promoter: a sequence upstream of the start of the gene that
encodes the RNA
2. RNA-coding sequence: the DNA sequence transcribed by RNA
polymerase into the RNA transcript.
3. Terminator: specifying where transcription stops.

Promoters:
DNA sequence to which RNA polymerase binds to
initiate transcription of a gene
20 to 200 bases
In bacteria, comparisons of promoter sequences of a
series of different genes isolated from E. coli showed:
• Transcription initiation site contains two sets of
sequences (similar in a variety of genes).
• Two common sequences (consensus sequences, 6
nucleotides).
• Consensus sequence TTGACA (position -35) in E.
coli and TATAAT (position -10)
• - 10 sequence (TATA box or Pribnow box) is
similar to sequences found at corresponding
positions in many eukaryotic promoters

Promoters of 10 different bacterial genes

Experimental evidence supports the functional
importance of the - 10 and - 35 promoter elements:
• Genes with promoter elements that differ from the
consensus sequences are transcribed less efficiently.
• Mutations induced in - 35 or - 10 consensus sequences
have strong effects on promoter function.
• Sites at which RNA polymerase binds to promoters
have been directly identified by footprinting
experiments (used to determine the sites at which
proteins bind to DNA).

DNA Footprinting:

Initiation of Transcription
Initiation of RNA chains involves three steps:
1.Binding of the RNA polymerase holoenzyme (α2, β, β′)
and σ to a promoter region in DNA.
2.Localized unwinding of the two strands of DNA by
RNA polymerase.
3.Formation of phosphodiester bonds between the first
few ribonucleotides in the nascent RNA chain.
4.Sigma factor σ is released and core enzyme remains
bound

Chain Elongation
•RNA polymerase then moves along the DNA template
strand, adding nucleotides to the 3′- end of the growing
RNA chain.
•After RNA polymerase has passed, the DNA strands
reform the duplex.
Chain Termination
RNA chain continues to grow until the RNA polymerase
encounters a termination sequence (signal).
•Rho-independent (Type I) terminators
• inverted repeat of a GC-rich sequence followed
by 6 or more A residues (common signal in E. coli)
•Rho-dependent (Type II) terminators
• Termination only in the presence of a protein
(helicase) called rho (p) to specific DNA sequences
for chain terminationDr. Reda Gaafar Associate Professor of Plant Molecular Genetics & Biotechnology

Chain Termination
Rho-independent (Type I) terminators
• RNA polymerase transcribes the terminator sequence
• RNA folds into a hairpin loop structure
• hairpin structure causes the RNA polymerase to slow
and stop
• String of U nucleotides downstream of the hairpin
destabilizes the pairing between the new RNA chain
and the DNA template strand.
• RNA polymerase dissociates from the template
Mutations disrupt the hairpin
partially or completely prevent
termination.

Transcription in eukaryotes
Prokaryotic mRNAs often contain the coding regions of 
two or more genes (multigenic mRNAs).
Eukaryotic transcripts that have been characterized
contain the coding region of a single gene (monogenic
mRNAs)
In eukaryotes, primary transcripts (pre-mRNAs) of
genes undergo three major modifications prior to
translation:
1.7-Methyl guanosine caps are added to the 5′- ends of
the primary transcripts.
2.Poly (A) tails are added to the 3′-ends of the
transcripts
3.Intron sequences are spliced out of transcripts.

3′- poly (A) tail is polyadenosine tract 20 to 200
nucleotides long.
Average half-life of a gene transcript
Eukaryotes is about five hours
E. Coli is less than five minutes
Eukaryotic RNA polymerases do not bind directly to
promoter sequences.
• There are several proteins (transcription factors)
required to initiate transcription.

Types of transcription factors:
• Basal (general) transcription factors
• involved in transcription from all polymerase II
promoters
• Each basal transcription factor is denoted
TFIIX (Transcription Factor X for RNA
polymerase II, where X is a letter identifying
the individual factor)
• Additional transcription factors
• involved in control of expression of individual
genes e.g. enhancers and silencers
• modulate the efficiency of initiation

Transcription by RNA Polymerase II
The promoters recognized by RNA polymerase II consist of short
conserved elements.
• They influence the efficiency of a promoter in initiating
transcription.
Structure of a promoter recognized by RNA polymerase II (the
promoter of thymidine kinase gene in mouse). .
Core promoter (TATA box + Inr)
Promoter-proximal elements (-50 to-200
nucleotides)

Eukaryotic genes transcribed by RNA polymerase II
have specific promoter sequences but…
No specific terminator sequences
Initiation of transcription
•Initiation of transcription involves the formation of
unwound DNA segment.
•Formation of unwound segment of DNA involves the
interaction of several transcription factors with
specific sequences in the promoter.

Initiation of
transcription by RNA
polymerase II
requires the
formation of a basal
transcription
initiation complex at
the promoter region.
Initiation of transcription
by polymerase II
TFIID has two subunits:
• TATA- binding protein (TBP) recognizes the
TATA box sequence
• Several other proteins called TBP-
associated factors (TAFs)

Transcription by RNA Polymerases I and III
RNA polymerases I and III initiate transcription by
processes similar and simpler than the one used by
polymerase I
Common transcription factor (TATA-binding protein
TBP) required for initiation of transcription by all 3
polymerases.
Promoters of genes transcribed by polymerases I and
III are quite different from those utilized by
polymerase II
• core sequence (-45 to +20)
• upstream control elements (-180 to -105)

RNA chain elongation and the addition of 5′-methyl
guanosine caps
•RNA chain elongation: by the same mechanism as the
RNA polymerases of prokaryotes.
•Early in the elongation process, 5′-ends of eukaryotic
pre-mRNAs are modified by the addition of 7-methyl
guanosine (7-MG) caps .

Pathway of biosynthesis of the 7-MG cap
Early stage in the transcription of a gene by RNA Polymerase II

Termination by chain cleavage and the addition of 3′
poly (A) tails
•3′ ends of RNA transcripts synthesized by RNA
polymerase II are produced by:
• endonucleolytic cleavage of the primary transcripts
rather than by the termination of transcription.
• The addition of poly (A) tails to eukaryotic mRNAs is
called polyadenylation
•formation of poly (A) tails on transcripts requires a
specific endonuclease (recognizes and binds to the
AAUAAA polyadenylation signal located 10 to 30
nucleotides upstream of the site of polyadenylation)
• Poly-A tails in most eukaryotes regulate translation and
contribute to mRNA stability.Dr. Reda Gaafar Associate Professor of Plant Molecular Genetics & Biotechnology

Concept Check 11.1
1. How did Griffith's experiments indicate the presence of a "transforming factor" in
bacteria?
2. What did Avery's experiments add to the knowledge gained from Griffith's
experiments?
3. Describe the experimental design that allowed Hershey and Chase to distinguish
between the two options for genetic material.

molecular genetics

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molecular genetics