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How to Study DNA
1. Genetic material
2. Expression product
DNA as Genetic Material
 DNA encodes all the information in the cell
 The composition of the DNA is the same in all cells
within an organism
– Variation among different cells is achieved by reading the
DNA differently
 DNA contains four bases that encode all the information
to make an organism’s life
DNA
 Meischer (1860’s)
DNA slightly acidic and composed of
large amounts of phosphorous and
nitrogen
 DNA consists of four kinds of bases
(A,C,G,T) joined to a sugar phosphate
backbone
 Bases carry the genetic information while
the phosphate backbone is structural
 Two complementary strands of bases (CG) and (A-T)
DNA primary structure
DNA (Deoxyribonucleic Acid)
a Polymer of Deoxyribonucleotide Units
Deoxyribonucleotide
Deoxy
Ribo
Nucleotide
DeoxyRibonucleotide
NH2
N
HC
O-
NH2
N
HC
N
CH
N
O
O
O
N
P O P O P OCH2 O
H
H
OOOH
H
HO
H
CH
N
N
HOCH2 O
H
H
H
HO
N
Deoxyadenosine
5´-triphosphate
(dATP)
H
H
DeoxyRibonucleoside Deoxyadenosine
DeoxyRibonucleotide
Phosphate
Group
O
O=P-O
O
5
CH2
O
N
C1
C4
Sugar
(Deoxyribose)
C3
C2
Nitrogenous base
(A, G, C, or T)
DeoxyRibonucleotide
5-carbon sugar (Deoxy ribose)
Nitrogenous base
Phosphate group
Deoxy ribo nucleotide
Ribose= Five Carbon Sugar Molecule
HOCH2 O
OH
5´
H
4´
H
3´
HO
H
1´
2´ H
OH
Ribose (RNA)
HOCH2 O
OH
5´
H
4´
H
3´
HO
H
1´
2´ H
H
Deoxyribose (DNA)
Backbone Sugar Molecules
NITROGEN BASES
It is composed of four different nitrogen bases
NH2
C
N
C
N
CH
9
C N
C
N
H
H
Adenine
HN
O C
O
C
1
N
H
C
C
O
C
Two
Purines
N
H N
C
CH
9
C N
C
N
H2 N
H
Guanine
CH3
H
Thymine
Two
Pyrimidines
O
NH2
C
H
N
C
C 1 C
N
H
H
Cytosine
Nitrogenous Bases
PURINES
Adenine (A)
2. Guanine (G)
1.
A or G
PYRIMIDINES
3. Thymine (T)
4. Cytosine (C)
T or C
BASE-PAIRINGS
Purines
Pyrimidines
Base
Pairs
Adenine (A)
Thymine (T)
A=T
Guanine (G)
Cytosine (C)
C G
# of
H-Bonds
2
3
3 H-bonds
G
C
BASE-PAIRINGS
H-bonds
G
C
T
A
Base Pairing Occurs
Through Hydrogen Bonds
G-C
A-T
Chargaff’s Rule
 Adenine must pair with Thymine
 Guanine must pair with Cytosine
 Their amounts in a given DNA molecule will be about
the same.
T
A
G
C
More of Chargaff’s Work
In any sample of DNA the
following is true:
– Amount of Cytosine =
Amount of Guanine
– Amount of Thymine =
Amount of Adenine
Polymerization of Nucleotides
3’ carbon
5’ carbon
Polynucleotides (Nucleic Acids)
5’ end
3’ end
The DNA Backbone is a
Deoxyribose Polymer
O
O P O
-
Deoxyribose sugars are linked by
Phosphodiester Bonds
O
OH
H2 C 5´ O
H
H
1´
H
-
O3´ 2´
H
H
O P O
O
OH
H2 C 5´ O
H
H
5´-p
1´
H
O3´ 2´H
O P O
O
H
OH
H2 C 5´ O
H
H
1´
H
3´
HO
2´
H
H
5´
3´-OH
3´
O
O P O
-
5´
O
5´
3´
H2 C 5´ O OH
H H
1´
H
-
3´ 2´
O
H
H
O P O
O
H2 C 5´ O OH
H H
1´
H
-
O3´ 2´
H
H
O P O
O
H2 C 5´ O OH
H H
1´
H
3´ 2´ H
HO H
3´
3´
5´
O
O P O
O
O P O
O
O
H2 C 5´ O OH
H
H 1´
H
3´ 2´
H
O
H
O P O
O
H2 C 5´ O OH
H
H 1´
H 3´ 2´ H
O
H
O P O
O
OH
H2 C 5´ O Base
H
H 1´
H
3´ 2´
H
O
H
O P O
O
OH
H2 C 5´ O Base
H
H 1´
H 3´ 2´ H
O
H
O P O
O
H2 C 5´ O OH
H
H 1´
OH
H2 C 5´ O Base
H
H 1´
H 3´ 2´ H
HO
H
H 3´ 2´ H
HO
H
O
O P O
O
C
CH3
HN
C
O
O C
C
N
H
H2 C
O
H
H
H
T
A
C
G
5´
3´
H
O
H NH2
O P O
C
H
N
C
O
O C
C
N
H
H2 C O
H
H
H
H
O
H O
O P O
C
CH3
HN
C
O
O C
C
H
H2 C O N
H
H
-
H
HO
H
H
T
A
3´
5´
G
C
=
A
T
Double-stranded DNA Forms
a Double Helix
DNA Double Helix
“Rungs of ladder”
Nitrogenous
Base (A,T,G or C)
“Legs of ladder”
Phosphate &
Sugar Backbone
DNA Double Helix
5
O
3
3
O
P
5
O
C
G
1
P
5
3
2
4
4
2
3
1
P
T
5
A
P
3
O
O
P
5
O
3
5
P
RIBO NUCLEIC ACID
A polymer composed of nucleotides that contain
the sugar ribose and one of the four bases cytosine,
adenine, guanine and uracile
Polynucleotide containing ribose sugar and uracile
instead of thymine
Genetic material of some viruses
Primary agent for transferring information from
the genome to the protein synthetic machinery
phosphate
group
URACIL
(U)
base with a single-ring
structure
sugar
(ribose)
Types of RNA
Three types of RNA:
a) messenger RNA (mRNA)
b) transfer RNA (tRNA)
c) ribosome RNA (rRNA)
 Remember:
All produced in the nucleus
A. Messenger RNA (mRNA)
 Carries the information for a specific protein.
 Made up of 500 to 1000 nucleotides long.
 Made up of codons (sequence of three bases: AUG methionine).
 Each codon is specific for an amino acid.
Codon
 There are 20
different possible
amino acids to make
from different
codons.
 3 possible stop
codon
 1 start codon
TAC on DNA
AUG on RNA
Codon Chart
Start Codon
Codon Chart
Messenger RNA (mRNA)
start
codon
mRNA
A U G G G C U C C A U C G G C G C A U A A
codon 1
protein
methionine
codon 2
codon 3
glycine
serine
codon 4
isoleucine
codon 5
codon 6
glycine
alanine
codon 7
stop
codon
Primary structure of a protein
aa1
aa2
aa3
peptide bonds
aa4
aa5
aa6
B. Transfer RNA (tRNA)
 Made up of 75 to 80 nucleotides long.
 Picks up the appropriate amino acid floating in the
cytoplasm (amino acid activating enzyme)
 Transports amino acids to the mRNA.
 Have anticodons that are complementary to mRNA
codons.
 Recognizes the appropriate codons on the mRNA and
bonds to them with H-bonds.
anticodon
codon in mRNA
anticodon
tRNA molecules
amino acid attachment site
amino
acid
amino acid
attachment
site
OH
The structure of transfer RNA (tRNA)
Transfer RNA (tRNA)
amino acid
attachment site
methionine
U A
C
anticodon
amino acid
C. Ribosomal RNA (rRNA)
 Made up of rRNA is 100 to 3000 nucleotides long.
 Important structural component of a ribosome.
 Associates with proteins to form ribosomes.
Ribosomes
 Large and small subunits.
 Composed of rRNA (40%) and proteins (60%).
 Both units come together and help bind the mRNA
and tRNA.
 Two sites for tRNA
a. P site (first and last tRNA will attach)
b. A site
Ribosomes
Origin
Cytosol
(eukaryotic
ribosome)
Chloroplasts
(prokaryotic
ribosome)
Complete
ribosome
80 S
Ribosomal
subunit
40 S
60 S
70 S
30 S
50 S
Mitochondrion 78 S
(prokaryotic
ribosome)
 30 S
 50 S
rRNA
components
18 S
5S
5.8 S
25 S
16 S
4.5 S
5 S
23 S
Proteins
18 S
5S
26 S
C. 33
C. 35
C.30
C.50
C. 24
C. 35
Ribosomes
Large
subunit
P
Site
A
Site
mRNA
A U G
Small subunit
C U A C U U C G
Study of Genetic Material
Number of chromosomes
Banding
Number of nucleotides
Sequencing
Structural genes
Cloning
Non-structural genes
Molecular marker
Central Dogma of Biology
DNA, RNA,
and the Flow of Information
Replication
Transcription
Translation
Central Dogma
(Modifications)
(2)Ribozymes
Transcription
DNA
RNA
Translation
Protein
(1) Reverse
transcription
Replication
(2)Self Replication (3)Self Replication
DNA Replication
1. Origin of Replication
2. Strand Separation
3. Priming
4. Synthesis of new strand DNA
DNA Replication
Origins of replication
1.
Replication Forks:
Hundreds of Y-shaped regions of replicating DNA
molecules where new strands are growing.
3’
5’
3’
Parental DNA Molecule
Replication
Fork
5’
DNA Replication
Origins of replication
2. Replication Bubbles:
Hundreds of replicating bubbles (eukaryotes).
Bubbles
Bubbles
DNA Replication
Strand Separation:
Unwinding and separation of the parental double helix
DNA
1. Helicase
Enzyme which catalyze the breaking H-Bonds between 2
nitrogen bases from different strand.
2. Single-Strand Binding Proteins
Proteins which attach and help keep the separated strands
apart.
DNA Replication
Strand Separation:
3. Topoisomerase
enzyme which relieves stress on the DNA molecule
by allowing free rotation around a single strand.
Enzyme
DNA
Enzyme
DNA Replication
Priming:
The attachment of complementary primer on the single stranded DNA
1.
RNA primers
Before new DNA strands can form, there must be small pre-existing
primers (RNA) present to start the addition of new nucleotides
(DNA Polymerase).
2.
Primase
Enzyme that polymerizes (synthesizes) the RNA Primer
DNA Replication
Synthesis of the new DNA Strands:
The additional of nucleotide on RNA primer
1. DNA Polymerase
with a RNA primer in place, DNA Polymerase (enzyme) catalyze
the synthesis of a new DNA strand in the 5’ to 3’ direction.
5’
3’
Nucleotide
DNA Polymerase
RNA
Primer
5’
DNA Replication
Synthesis of the new DNA Strands
2. Leading Strand
synthesized as a single polymer in the 5’ to 3’ direction.
5’
3’
5’
Nucleotides
DNA Polymerase
RNA
Primer
DNA Replication
Synthesis of the new DNA Strands
3. Lagging Strand
It also synthesized in the 5’ to 3’ direction, but
discontinuously against overall direction of replication.
Leading Strand
5
’
3’
DNA Polymerase
RNA Primer
3’
5’
5’
3’
3’
5’
Lagging Strand
DNA Replication
Synthesis of the new DNA Strands
4. Okazaki Fragment
series of short segments on the lagging strand.
DNA
Polymerase
Okazaki Fragment
RNA
Primer
5’
3’
Lagging Strand
3’
5’
DNA Replication
Synthesis of the new DNA Strands
5. DNA ligase
a linking enzyme that catalyzes the formation of a covalent
bond from the 3’ to 5’ end of joining stands.
Example: joining two Okazaki fragments together.
DNA ligase
5’
3’
Okazaki Fragment 1
Lagging Strand
Okazaki Fragment 2
3’
5’
DNA Replication
Synthesis of the new DNA Strands
6. Proofreading
initial base-pairing errors are usually corrected by DNA
polymerase.
DNA Replication
Semiconservative Model
Watson and Crick
the two strands of the parental molecule separate, and each
functions as a template for synthesis of a new
complementary strand.
DNA Template
Parental DNA
New DNA
DNA Repair
Excision repair
1. Damaged segment is excised by a repair enzyme (there
are over 50 repair enzymes).
2. DNA polymerase and DNA ligase replace and bond the
new nucleotides together.
What is gene expression?
The activation of a gene that results in a protein.
Biological processes, such as transcription, and in
case of proteins, also translation, that yield a gene
product.
 A gene is expressed when its biological product is
present and active.
 Gene expression is regulated at multiple levels.
Expression of Genetic Information
Production of proteins requires two steps:
Transcription involves an enzyme (RNA polymerase)
making an RNA copy of part of one DNA strand.
There are four main classes of RNA:
i. Messenger RNAs (mRNA), which specify the amino acid
sequence of a protein by using codons of the genetic code.
ii. Transfer RNAs (tRNA).
iii. Ribosomal RNAs (rRNA).
 Translation converts the information in mRNA into the
amino acid sequence of a protein using ribosomes, large
complexes of rRNAs and proteins.
Steps of gene expression
 Transcription –
DNA is read to
make a mRNA in
the nucleus of
cells
 Translation –
Reading the
mRNA to make a
protein in the
cytoplasm
Three (3) regulatory elements
of transcription
 Coding region:
DNA that code for a specific polypeptide
(protein)
 Promoter :
DNA segment that recognizes RNA polymerase
 Operator :
Element that serves as a binding site for an inhibitor
protein (modulator) that controls transcription
Promoter Region on DNA
 Upstream from transcription start site
 Initial binding site of RNA polymerase and initiation factors (IFs)
 Promoter recognition: a prerequisite for initiation
Prokaryotic promoter regions
-35 site = TTGACA
-10 site: “TATA” box
66
Promoter Region on DNA
(TATA box)
Pol II Eukaryotic
Promoter Elements
GC box
~200 bp
CCAAT box
~100 bp
TATA box
~30 bp
Transcription
start site (TSS)
Pol II Eukaryotic
Promoter Elements
Cap Region/Signal:
nCAGTnG
TATA box: (~ 25 bp upstream)
TATAAAnGCCC
CCAAT box: (~100 bp upstream)
TAGCCAATG
GC box: (~200 bp upstream)
A T A G G C G nGA
Prokaryotic and eukaryotic gene
organization
Prokaryotic
transcriptional
regulatory regions
(promoters and
operators) lie close to
the transcription start
site
Functionally related
genes are frequently
located near each other
These “operons” are
transcribed into a
single mRNA with
internal translation
initiation sites
Prokaryotic Gene Expression
Expression mainly by controlling transcription
Promoter
Cistron1
Cistron2 CistronN Terminator
Transcription
RNA Polymerase
mRNA 5’
3’
1
2
Translation
C
N
N
N
Ribosome, tRNAs,
Protein Factors
C
N
C
1
2
Polypeptides
3
Operons
Genes that work together are located together
 A promoter plus a set of adjacent genes whose gene
products function together.
They are controlled as a unit
They usually contain 2 –6 genes (up to 20 genes)
These genes are transcribed as a polycistronic transcript.
 It is relatively common in prokaryotes
 It is rare in eukaryotes
Operon System
The lactose (lac) operon
Pi
I
Q3
P
Q1
Z
Q2
Y
• Contains several elements
–
–
–
–
lacZ gene = β-galactosidase
lacY gene = galactosidase permease
lacA gene = thiogalactoside transacetylase
lacI gene = lac repressor
–
–
–
–
Pi = promoter for the lacI gene
P = promoter for lac-operon
Q1 = main operator
Q2 and Q3 = secondary operator sites (pseudo-operators)
A
Regulation of the lac operon
Pi
I
Q3
P
Q1
Z
Q2
LacZ
lacI repressor
Y
LacY
Inducer molecules→ Allolactose:
- natural inducer, degradable IPTG
(Isopropylthiogalactoside)
- synthetic inducer, not metabolized
A
LacA
The lac operon:
model for gene expression
Includes three protein
synthesis coding region-sometimes called "genes" as
well as region of
chromosome that controls
transcription of genes
Genes for proteins involved
in the catabolism or
breakdown of lactose
When lactose is absent, no
transcription of gene since
no need for these proteins
When lactose is present,
transcription of genes takes
place so proteins are
available to catalyze
breakdown of lactose
Eukaryotic gene
Eukaryotic gene Expression
1.Transcripts begin and end
beyond the coding region
2.The primary transcript is
processed by:
5’ capping
3’ formation / polyA
splicing
3.Mature transcripts are
transported to the
cytoplasm for translation
Regulation of gene expression
Promoter
1. DNA replication
Gene (red) with an intron (green)
Plasmid
single copy vs. multicopy plasmids
2. Transcription
3. Posttranscriptional
processing
4. Translation
5. Posttranslational
processing
Primary
transcript
mRNA degradation
Mature
mRNA
inactive
protein
active
protein
Protein degradation
Regulation of gene expression
 Gene expression is regulated—not all genes are constantly
active and having their protein produced
 The regulation or feedback on gene expression is how the
cell’s metabolism is controlled.
 This regulation can happen in different ways:
1. Transcriptional control (in nucleus):
e.g. chromatin density and transcription factors
2. Posttranscriptional control (nucleus)
e.g. mRNA processing
3. Translational control (cytoplasm)
e.g. Differential ability of mRNA to bind ribosomes
4. Posttranslational control (cytoplasm)
e.g. changes to the protein to make it functional
 When regulation of gene expression goes wrong—cancer!
1. Transcription control
Eukaryotic gene expression
Gene regulation of the transcription
Condition 2
1
Chr. I
1
10
Chr. II
Chr. III
2
19
“turned “turned
“turned
off”
off”
on”
on”
4
5
6
7
8
3
11
12
20 21
22
constitutively
expressed gene
13 14 15 16
23
induced
gene
24
9
17
25
18
26
repressed
gene
inducible/ repressible genes
Gene regulation
upregulated
gene expression
1
2
10
19
Condition 43
down regulated
gene expression
3
4
11
12
20 21
22
constitutively
expressed gene
5
7
8
13 14 15 16
17
23
6
24
25
9
18
26
Definitions
Constitutively expressed genes
Genes that are actively transcribed (and translated) under all
experimental conditions, at essentially all developmental stages, or in
virtually all cells.
Inducible genes
Genes that are transcribed and translated at higher levels in response
to an inducing factor
Repressible genes
Genes whose transcription and translation decreases in response to a
repressing signal
Housekeeping genes
–genes for enzymes of central metabolic pathways (e.g. TCA cycle)
–these genes are constitutively expressed
–the level of gene expression may vary
2. Post-Transcriptional Modification in
Eukaryotes
 Primary transcript formed first
 Then processed (3 steps) to form mature mRNA
 Then transported to cytoplasm
Step 1: 7- methyl-guanosine “5’-cap”
added to 5’ end
Step 2: introns spliced out; exons link
up
Step 3: Poly-A tail added to 3’ end
mature mRNA
5’-cap- exons -3’ PolyA tail
Intron Splicing in Eukaryotes
• Exons : coding regions
• Introns : noncoding regions
• Introns are removed by “splicing”
GU at 5’ end
of intron
AG at 3’ end
of intron
88
Splicesomes Roles in Splicing out Intron
RNA splicing occurs in small nuclear ribonucleoprotein
particles (snRNPS) in spliceosomes
89
Splicesomes Roles in Splicing out Intron
 5’ exon then moves to the 3’ splice acceptor site where a
second cut is made by the spliceosome
 Exon termini are joined and sealed
1
2
1
2
1
2
90
Translation
Three parts:
1. Initiation: start codon (AUG)
2. Elongation:
3. Termination: stop codon (UAG)
Translation
Large
subunit
P
Site
A
Site
mRNA
A U G
Small subunit
C U A C U U C G
Initiation
aa1
aa2
2-tRNA
1-tRNA
anticodon
hydrogen
bonds
U A C
A U G
codon
G A U
C U A C U U C G A
mRNA
peptide bond
aa3
aa1
aa2
3-tRNA
1-tRNA
anticodon
hydrogen
bonds
U A C
A U G
codon
2-tRNA
G A A
G A U
C U A C U U C G A
mRNA
aa1
peptide bond
aa3
aa2
1-tRNA
3-tRNA
U A C
(leaves)
2-tRNA
A U G
G A A
G A U
C U A C U U C G A
mRNA
Ribosomes move over one codon
aa1
peptide bonds
aa4
aa2
aa3
4-tRNA
2-tRNA
A U G
3-tRNA
G C U
G A U G A A
C U A C U U C G A A C U
mRNA
aa1
peptide bonds
aa4
aa2
aa3
2-tRNA
4-tRNA
G A U
(leaves)
3-tRNA
A U G
G C U
G A A
C U A C U U C G A A C U
mRNA
Ribosomes move over one codon
aa1
peptide bonds
aa5
aa2
aa3
aa4
5-tRNA
U G A
3-tRNA
4-tRNA
G A A G C U
G C U A C U U C G A A C U
mRNA
peptide bonds
aa1
aa5
aa2
aa3
aa4
5-tRNA
U G A
3-tRNA
G A A
4-tRNA
G C U
G C U A C U U C G A A C U
mRNA
Ribosomes move over one codon
aa4
aa5
Termination
aa199
aa3 primary
structure
of
a
protein
aa2
aa200
aa1
200-tRNA
A C U
mRNA
terminator
or stop
codon
C A U G U U U A G
Translation
Ribosome
Amino Acids forming
Peptide chain
P Site
Met
His
Tyr
A Site
Val
Pro
3’
E Site
tRNA
anti-codon
5’
codon
AUG
CAU
GGA
UAC
GUA
CCU
mRNA strand
Translation
The difference
• Eukaryotic and prokaryotic translation can react differently to
certain antibiotics
Puromycin
an analog tRNA and a general inhibitor of protein synthesis
 Cycloheximide
only inhibits protein synthesis by eukaryotic ribosomes
 Chloramphenicol, Tetracycline, Streptomycin
inhibit protein synthesis by prokaryotic ribosome
End Product
 The end products of protein synthesis is a primary
structure of a protein.
 A sequence of amino acid bonded together by peptide
bonds.
aa2
aa1
aa3
aa4
aa5
aa199
aa200
Polyribosome
Groups of ribosomes reading same mRNA simultaneously
producing many proteins (polypeptides).
incoming
large
subunit
1
incoming
small subunit
2
3
4
polypeptide
5
6
7
mRNA
Prokaryotes vs eukaryotes:
key points
Prokaryotes
Eukaryotes
Operons
(functional grouping)
Monocistronic RNAs
(One mRNA, one protein)
Polycistronic mRNAs
(single mRNA, multiple ORFs)
Ribosome scanning
No splicing
Regulatory sequences lie
near (~100 bp) the start site
Translation is concurrent
with transcription
Often spliced
Regulatory sequences can be
far (>1 kb) from the start site
RNA processing is concurrent
with transcription; translation
occurs in a separate
compartment
TYPES OF PROTEINS
Enzymes (Helicase)
Carrier (Haemoglobine)
Immunoglobulin (Antibodies)
Hormones (Steroids)
Structural (Muscle)
Ionic (K+,Na+)
Coupled transcription and translation in bacteria
original
base triplet
in a DNA
strand
As DNA is replicated, proofreading
enzymes detect the mistake and
make a substitution for it:
a base
substitution
within the
triplet (red)
POSSIBLE OUTCOMES:
OR
One DNA molecule
carries the original,
unmutated sequence
VALINE
PROLINE
The other DNA
molecule carries
a gene mutation
THREONINE
VALINE
LEUCINE
HISTIDINE
GLUTAMATE
A summary of transcription and translation in a eukaryotic cell