Download DNA Replication - Gadjah Mada University

How to Study
Genome
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
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 (C-G) and (A-T)
DNA (Deoxyribonucleic Acid)
a Polymer of Deoxyribonucleotide Units
Deoxyribonucleotide
Deoxy
Ribo
Nucleotide
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
Nucleotide
Phosphate group
5-carbon sugar
Nitrogenous base
Nucleotide
Phosphate
Group
O
O=P-O
O
5
CH2
O
N
C1
C4
Sugar
(deoxyribose)
C3
C2
Nitrogenous base
(A, G, C, or T)
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
DNA
It is Composed of Four Different Ribonucleotides
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
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
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
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
RNA
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 uracil
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.
A. 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
rRNA
components
18 S
5S
5.8 S
25 S
Proteins
70 S
30 S
50 S
16 S
4.5 S
5 S
23 S
C. 24
C. 35
 30 S
 50 S
18 S
5S
26 S
C. 33
C. 35
Mitochondrion 78 S
(prokaryotic
ribosome)
C.30
C.50
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.
5’
3’
Replication Forks:
hundreds of Y-shaped regions of replicating DNA
molecules where new strands are growing.
Parental DNA Molecule
3’
Replication
Fork
5’
DNA Replication
Origins of replication
2. Replication Bubbles:
a.Hundreds of replicating bubbles (Eukaryotes).
b.Single replication fork (bacteria).
Bubbles
Bubbles
DNA Replication
Strand Separation:
1. Helicase
Enzyme which catalyze the unwinding and separation
(breaking H-Bonds) of the parental double helix.
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
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
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.
Gene Expression
Transcription
Translation
What is gene expression?
 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
Beadle and Tatum (1941) showed in the fungus
Neurospora crassa that there is a relationship
between a gene and each enzyme needed in a
biochemical pathway, resulting in the one geneone enzyme hypothesis
(now modified to one gene-one polypeptide, since
not all proteins are enzymes and some require
more than one polypeptide).
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).
iv. Small nuclear RNAs (snRNA), found only in eukaryotes.
 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 our
cells
 Translation –
Reading the
mRNA to make a
protein in the
cytoplasm
mRNA
Synthesis
 DNA template: 3’-to-5’
 RNA synthesis: 5’-3’; no primer needed
59
Expression of Genetic
Information
 Only some of the genes in a cell are active at any given
time, and activity also varies by tissue type and
developmental stage.
 Regulation of gene expression is not completely
understood, but it has been shown to involve an array
of controlling signals.
a. Jacob and Monod (1961) proposed the operon model to
explain prokaryotic gene regulation, showing that a genetic
switch is used to control production of the enzymes needed
to metabolize lactose. Similar systems control many genes in
bacteria and their viruses.
b. Genetic switches used in eukaryotes are different and more
complex, with much remaining to be learned about their
function.
Prokaryotic 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
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
a promoter plus a set of adjacent genes whose gene
products function together.
usually contain 2 –6 genes, (up to 20 genes)
these genes are transcribed as a polycistronic transcript.
 relatively common in prokaryotes
 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
Eukaryotic gene expression
Eukaryotic gene Expression
 Transcripts begin and end beyond the coding region (5’UTR and
3’UTR)
 The primary transcript is processed by:
a. 5’ capping
b. 3’ formation polyA
c. splicing
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
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
Modulators of transcription
 Modulators:
(1) specificity factors, (2) repressors, (3) activators
1. Specificity factors:
Alter the specificity of RNA polymerase
Examples: σ-factors (s70, s32 )
s70
Standard
promoter
s32
Housekeeping gene
Heat shock
promoter
Heat shock gene
Modulators of transcription
2. Repressors:





mediate negative gene regulation
may impede access of RNA polymerase to the promoter
actively block transcription
bind to specific “operator” sequences (repressor binding sites)
Repressor binding is modulated by specific effectors
Effector
(e.g. endproduct)
Repressor
Operator
Promoter
Coding sequence
Negative regulation
Repressor
Effector
Example:
lac operon
RESULT:
Transcription occurs
when the gene is
derepressed
Negative regulation
Repressor
Effector (= co-repressor)
Example:
pur-repressor in E. coli;
regulates transcription of
genes involved in nucleotide
metabolism
Modulators of transcription
3. Activators:
 mediate positive gene regulation
 bind to specific regulatory DNA sequences (e.g. enhancers)
 enhance the RNA polymerase -promoter interaction and actively
stimulate transcription
 common in eukaryotes
Activator
RNA pol.
promoter
Coding sequence
Positive regulation
Activator
RNA polymerase
Positive regulation
Activator
Effector
RNA polymerase
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
81
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
82
Splicesomes Roles in Splicing out Intron
RNA splicing occurs in small nuclear ribonucleoprotein
particles (snRNPS) in spliceosomes
83
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
84
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
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